CN115197307A - Protein IbGER5 for regulating and controlling plant stress resistance and coding gene and application thereof - Google Patents

Abstract

The invention discloses a protein IbGER5 regulating plant stress resistance and its encoding gene and application. Specifically disclosed are proteins whose amino acid sequence is SEQ ID No. 1, related biological materials, and applications thereof in regulating plant salt tolerance and/or drought resistance. In the present invention, the IbGER5 gene derived from sweet potato is introduced into the receptor control chestnut incense, and the transgenic sweet potato plants that overexpress and interfere with the expression of the IbGER5 gene are obtained. The salt tolerance and drought resistance of the transgenic sweetpotato plants overexpressing the IbGER5 gene were significantly reduced, and the salt tolerance and drought resistance of the transgenic sweetpotato plants that interfered with the expression of the IbGER5 gene (RNAi) were significantly improved, indicating that the IbGER5 protein and its encoding gene provided by the present invention have The function of regulating plant salt tolerance and/or drought resistance can be widely used in sweet potato anti-reversal gene breeding.

Description

Protein IbGER5 Regulating Plant Stress Resistance and Its Encoding Gene and Application

technical field

The invention belongs to the field of biotechnology, and particularly relates to a protein IbGER5 regulating plant stress resistance and its encoding gene and application.

Background technique

Sweet potato (Ipomoea batatas (L.) Lam.) is an important food, fodder and industrial fuel crop with both health care and medicinal value, and is widely grown in all parts of my country. With the continuous reduction of arable land, sweet potatoes are mainly planted in marginal land such as drought or salinization, while the salinity of salinized land in my country is 0.6-10%. The shortage has seriously affected the normal growth of crops, and brought great difficulties to the further development of sweet potato production. Therefore, cultivating new high-quality sweet potato varieties with strong salt and drought resistance has become one of the important measures to promote the development of the sweet potato industry.

Although conventional sweetpotato breeding technology has played an important role in variety improvement, due to the problems of self-incompatibility, unstable hybrid progeny, lack of germplasm resources, and long breeding cycle of sweetpotato, traditional hybrid breeding methods are difficult to breed. New sweet potato varieties with strong salt and drought resistance, and the use of genetic engineering methods can overcome the obstacles of species isolation and gene linkage in conventional breeding, and can improve sweet potato traits at the molecular level. This approach has potential to promote the breeding and production of sweet potato varieties. Mining important salt- and drought-resistant genetic resources plays a key role in cultivating new varieties of salt- and drought-resistant plants. The development and utilization of salt- and drought-resistant plants have immeasurable ecological, economic and social benefits.

SUMMARY OF THE INVENTION

The technical problem to be solved by the present invention is how to regulate the stress resistance of plants (such as sweet potato). The technical problem to be solved is not limited to the described technical subject, and other technical subjects not mentioned herein can be clearly understood by those skilled in the art through the following description.

In order to solve the above-mentioned technical problems, the present invention firstly provides the application of protein or substances regulating the activity and/or content of the protein, and the application can be any of the following:

D1) the application of protein or substances regulating the activity and/or content of the protein in regulating plant stress resistance;

D2) the application of protein or substances regulating the activity and/or content of the protein in the preparation of products for regulating plant stress resistance;

D3) application of protein or substances regulating the activity and/or content of said protein in cultivating stress-resistant plants;

D4) the application of protein or substances regulating the activity and/or content of the protein in the preparation of products for cultivating stress-resistant plants;

D5) Application of protein or substances regulating the activity and/or content of said protein in plant breeding;

The protein name is IbGER5 and can be any of the following:

A1) the amino acid sequence is the protein of SEQ ID No. 1;

A2) A protein with more than 80% identity and the same function as the protein shown in A1) obtained by substituting and/or deleting and/or adding amino acid residues to the amino acid sequence shown in SEQ ID No. 1;

A3) A fusion protein with the same function obtained by linking a tag to the N-terminus and/or C-terminus of A1) or A2).

In order to facilitate purification or detection of the protein in A1), a tag protein can be attached to the amino terminus or carboxyl terminus of the protein consisting of the amino acid sequence shown in SEQ ID No. 1 in the sequence listing.

The tagged proteins include but are not limited to: GST (glutathione sulfhydryl transferase) tagged protein, His6 tagged protein (His-tag), MBP (maltose binding protein) tagged protein, Flag tagged protein, SUMO tagged protein, HA tag protein, Myc-tagged protein, eGFP (enhanced green fluorescent protein), eCFP (enhanced cyan fluorescent protein), eYFP (enhanced yellow-green fluorescent protein), mCherry (monomeric red fluorescent protein), or AviTag-tagged protein.

Those of ordinary skill in the art can easily mutate the nucleotide sequence encoding the protein IbGER5 of the present invention by known methods, such as directed evolution or point mutation. Those artificially modified nucleotides with 75% or more identity to the nucleotide sequence of the protein IbGER5 isolated by the present invention, as long as they encode the protein IbGER5 and have the function of the protein IbGER5, are derived from the present invention Nucleotide sequences and are equivalent to the sequences of the present invention.

The above-mentioned 75% or more identity may be 80%, 85%, 90% or more than 95% identity.

Herein, identity refers to the identity of amino acid sequences or nucleotide sequences. Amino acid sequence identity can be determined using homology search sites on the Internet, such as the BLAST page of the NCBI homepage website. For example, in advanced BLAST2.1, by using blastp as the program, set the Expect value to 10, set all Filters to OFF, use BLOSUM62 as the Matrix, and set the Gap existence cost, Per residue gap cost and Lambda ratio to be respectively 11, 1 and 0.85 (default value) and performing a search to calculate the identity of the amino acid sequence, the value (%) of the identity can then be obtained.

Herein, the identity of more than 80% may be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

Herein, the substance that modulates the activity and/or content of the protein may be a substance that modulates the expression of a gene encoding the protein IbGER5.

In the above, the substance that regulates gene expression may be a substance that performs at least one of the following 6 kinds of regulation: 1) regulation at the level of the gene transcription; 2) regulation after the gene is transcribed ( That is, the regulation of splicing or processing of the primary transcript of the gene); 3) the regulation of the RNA transport of the gene (that is, the regulation of the mRNA transport of the gene from the nucleus to the cytoplasm); 4) regulation of the translation of the gene; 5) regulation of the mRNA degradation of the gene; 6) post-translation regulation of the gene (that is, regulation of the activity of the protein translated by the gene) ).

The substance that regulates gene expression can specifically be any of the biological materials described in the following B1)-B3).

Further, the substance that regulates gene expression may be a substance (including nucleic acid molecules or vectors) that increases or up-regulates the expression of the gene encoding the protein IbGER5.

Further, the substance that regulates gene expression can also be a substance (including nucleic acid molecule or vector) that inhibits or reduces or down-regulates the expression of the gene encoding the protein IbGER5.

In the above application, the protein IbGER5 can be derived from sweet potato (Ipomoea batatas (L.) Lam.).

Further, the protein IbGER5 may be a protein IbGER5 that regulates plant stress resistance.

Further, the protein IbGER5 can be the protein IbGER5 that regulates the salt tolerance and drought resistance of plants.

The present invention also provides the application of the biological material related to the protein IbGER5, and the application can be any of the following:

E1) Application of the biological material related to the protein IbGER5 in regulating plant stress resistance;

E2) application of the biological material related to the protein IbGER5 in the preparation of a product for regulating plant stress resistance;

E3) application of the biological material related to the protein IbGER5 in cultivating stress-resistant plants;

E4) application of the biological material related to the protein IbGER5 in the preparation of products for cultivating stress-resistant plants;

E5) the application of the biological material related to the protein IbGER5 in plant breeding;

The biological material may be any of the following B1) to B8):

B1) a nucleic acid molecule encoding the protein of claim 1 or 2;

B2) a nucleic acid molecule that inhibits or reduces the expression of a gene encoding the protein of claim 1 or 2;

B3) an expression cassette containing the nucleic acid molecule of B1) and/or B2);

B4) a recombinant vector containing the nucleic acid molecule described in B1) and/or B2), or a recombinant vector containing the expression cassette described in B3);

B5) a recombinant microorganism containing the nucleic acid molecule described in B1) and/or B2), or a recombinant microorganism containing the expression cassette described in B3), or a recombinant microorganism containing the recombinant vector described in B4);

B6) a transgenic plant cell line comprising the nucleic acid molecule described in B1) and/or B2), or a transgenic plant cell line comprising the expression cassette described in B3), or a transgenic plant cell line comprising the recombinant vector described in B4);

B7) a transgenic plant tissue containing the nucleic acid molecule of B1) and/or B2), or a transgenic plant tissue containing the expression cassette of B3);

B8) A transgenic plant organ containing the nucleic acid molecule of B1) and/or B2), or a transgenic plant organ containing the expression cassette of B3).

In the above-mentioned application, the nucleic acid molecule can be any of the following:

C1) the coding sequence is the DNA molecule shown in SEQ ID No.2 or positions 1-204 of SEQ ID No.2;

C2) The nucleotide sequence is the DNA molecule shown in SEQ ID No. 2 or positions 1-204 of SEQ ID No. 2.

The DNA molecule shown in SEQ ID No. 2 (the IbGER5 gene) encodes the amino acid sequence of the protein IbGER5 of SEQ ID No. 1.

The nucleotide sequence shown in SEQ ID NO. 2 is the nucleotide sequence of the protein IbGER5 encoding gene (CDS).

B1) The nucleic acid molecule may also include a nucleic acid molecule obtained by codon bias modification on the basis of the nucleotide sequence shown in SEQ ID No. 2.

B1) The nucleic acid molecules also include nucleic acid molecules that are more than 95% identical to the nucleotide sequence shown in SEQ ID No. 2 and originate from the same species.

The protein IbGER5 gene (IbGER5 gene) of the present invention can be any nucleotide sequence capable of encoding the protein IbGER5. Considering the degeneracy of codons and the codon preferences of different species, those skilled in the art can use codons suitable for expression of a particular species as needed.

The expression cassette comprises a promoter, a nucleic acid molecule encoding the protein IbGER5 and a terminator, the promoter can be a CaMV35S promoter, a NOS promoter or an OCS promoter, and the terminator can be a NOS terminator or OCS polyA terminator.

The nucleic acid molecule described herein can be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecule can also be RNA, such as gRNA, mRNA, siRNA, shRNA, sgRNA, miRNA, or antisense RNA.

The vectors described herein are well known to those skilled in the art and include, but are not limited to, plasmids, phages (eg, lambda phage or M13 filamentous phage, etc.), cosmids (ie, cosmids), Ti plasmids, or viral vectors. Specifically, it can be pMD19-T vector and/or pCAMBIA1300 vector and/or pFGC5941 vector.

The recombinant expression vector containing the IbGER5 gene can be constructed using the existing plant expression vector. The plant expression vectors include, but are not limited to, such as binary Agrobacterium vectors and vectors that can be used for plant microprojectile bombardment, and the like. The plant expression vector may also contain the 3' untranslated region of the exogenous gene, ie, containing the polyadenylation signal and any other DNA fragments involved in mRNA processing or gene expression. The polyadenylation signal can guide the addition of polyadenylic acid to the 3' end of the mRNA precursor, such as including but not limited to Agrobacterium crown gall-inducing (Ti) plasmid genes (such as nopaline synthase Nos gene), plant genes (eg soybean storage protein gene) 3'-terminal transcribed untranslated regions have similar functions.

When using the IbGER5 gene to construct a recombinant plant expression vector, any enhanced promoter or constitutive promoter can be added before its transcription initiation nucleotide, including but not limited to cauliflower mosaic virus (CaMV) 35S promoter , maize ubiquitin promoter (ubiquitin), they can be used alone or in combination with other plant promoters; in addition, when using the gene of the present invention to construct plant expression vectors, enhancers can also be used, including translation enhancers or transcription enhancers These enhancer regions can be ATG start codons or adjacent region start codons, etc., but must be the same as the reading frame of the coding sequence to ensure the correct translation of the entire sequence. The translation control signals and initiation codons can be derived from a wide variety of sources, either natural or synthetic. The translation initiation region can be derived from a transcription initiation region or a structural gene.

In order to facilitate the identification and screening of transgenic plant cells or plants, the plant expression vector used can be processed, such as adding, including but not limited to, genes that can be expressed in plants and encode enzymes or light-emitting compounds that can produce color changes (GUS gene, luciferase gene, etc.), antibiotic markers with resistance (gentamicin marker, kanamycin marker, etc.) or chemical reagent-resistant marker genes (such as herbicide-resistant genes) and the like. Considering the safety of transgenic plants, the transformed plants can be directly screened under stress without adding any selectable marker gene.

Using any vector that can guide the expression of exogenous genes in plants, the IbGER5 gene or gene fragment provided by the present invention is introduced into plant cells or recipient plants to obtain transgenic plants with altered stress resistance. The expression vector carrying the IbGER5 gene can transform plant cells or tissues by using conventional biological methods such as Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, electrical conductivity, and Agrobacterium-mediated transformation, and transform the transformed plant tissue. Cultivated into plants.

The microorganisms described herein can be yeast, bacteria, algae or fungi. Among them, the bacteria can be from Escherichia, Erwinia, Agrobacterium, Flavobacterium, Alcaligenes, Pseudomonas (Pseudomonas), Bacillus, etc. Specifically, it can be Escherichia coli DH5α and/or Agrobacterium tumefaciens EHA105.

The recombinant vector can specifically be the recombinant vector pCAMBIA1300-GFP-IbGER5 and/or pFGC5941-IbGER5.

The recombinant vector pCAMBIA1300-GFP-IbGER5 is to replace the fragment (small fragment) between the KpnI and SalI recognition sites of the pCAMBIA1300-GFP vector with a DNA fragment whose nucleotide sequence is SEQ ID No. 2 in the sequence listing, maintaining pCAMBIA1300 -The other sequences of the GFP vector remain unchanged, and the obtained recombinant expression vector. The recombinant vector pCAMBIA1300-GFP-IbGER5 expresses the protein IbGER5 shown in SEQ ID No. 1 in the sequence listing. The recombinant vector pCAMBIA1300-GFP-IbGER5 has an expression cassette, and the nucleotide sequence of the expression cassette includes the CaMV35S promoter, the gene encoding IbGER5 protein (SEQ ID No. 2) and the gene encoding green fluorescent protein (GFP).

The pCAMBIA1300-GFP vector is based on pCAMBIA1300. The CaMV 35S fragment is inserted between the EcoRI and SacI recognition sites of the pCAMBIA1300 vector, and the GFP fragment is inserted between the SalI and PstI recognition sites of the pCAMBIA1300 vector.

The recombinant vector pFGC5941-IbGER5 is to replace the small fragment between the XhoI and SwaI recognition sequences of the pFGC5941 vector with the DNA molecule fragment shown in positions 1 to 204 from the 5' end of SEQ ID No.2 in the sequence listing. The fragment (small fragment) between the recognition sites of the endonucleases BamHI and XbaI is replaced with the nucleotide sequence that is the reverse of the DNA molecule shown in SEQ ID No. 2 from the 5' end in the Sequence Listing from positions 1 to 204 Complementary sequence, keeping other sequences of pFGC5941 vector unchanged, the obtained recombinant expression vector. The recombinant vector pFGC5941-IbGER5 interferes with the expression of the protein IbGER5 shown in SEQ ID No. 1 in the sequence listing. The recombinant vector pFGC5941-IbGER5 has an expression cassette, and the nucleotide sequence of the expression cassette contains the CaMV35S promoter, the gene fragment encoding the IbGER5 protein and the OCS polyA terminator. The recombinant microorganism can be obtained by introducing the recombinant vector into the starting microorganism.

The recombinant microorganism can specifically be recombinant Agrobacterium EHA105/pCAMBIA1300-GFP-IbGER5 and/or EHA105/pFGC5941-IbGER5.

The recombinant Agrobacterium EHA105/pCAMBIA1300-GFP-IbGER5 is a recombinant bacterium obtained by introducing the recombinant vector pCAMBIA1300-GFP-IbGER5 into Agrobacterium tumefaciens EHA105.

The recombinant Agrobacterium EHA105/pFGC5941-IbGER5 is a recombinant bacterium obtained by introducing the recombinant vector pFGC5941-IbGER5 into Agrobacterium tumefaciens EHA105.

The present invention also provides a method for cultivating a transgenic plant, the method comprising increasing and/or reducing the content and/or activity of the protein IbGER5 in the target plant to obtain the transgenic plant.

In the above method, increasing the content and/or activity of the protein IbGER5 in the target plant can be achieved by increasing the expression level of the gene encoding the protein IbGER5 in the target plant.

In the above method, increasing the expression level of the gene encoding the protein IbGER5 in the target plant can be achieved by introducing the gene encoding the protein IbGER5 into the target plant.

In the above method, the reducing the content and/or activity of the protein IbGER5 in the target plant is achieved by reducing the expression level of the gene encoding the protein IbGER5 in the target plant.

In the above method, the reducing the expression level of the gene encoding the protein IbGER5 in the target plant may be the use of gene mutation, gene knockout, gene editing or gene knockdown technology to make the gene encoding the protein IbGER5 in the target plant genome active. decline or inactivation.

Further, the use of gene knockout technology to reduce the activity of the encoding gene of the protein IbGER5 in the target plant genome or inactivate can be carried out by using an RNA interference vector, and the RNA interference vector contains the nucleotide sequence of SEQID No.2. The DNA molecule shown at positions 1-204.

In the above method, the transgenic plant may be a plant with altered stress resistance, further, the plant with altered stress resistance may be reduced (down-regulated) and/or stress resistance (such as salt tolerance and/or drought resistance) or increased (up-regulated) transgenic plants.

The transgenic plants with reduced stress resistance are transgenic plants with lower stress resistance than the plants of interest.

The transgenic plant with improved stress resistance is a transgenic plant (stress resistant plant) having a higher stress resistance than the target plant.

In the above method, the encoding gene of the protein IbGER5 can be any of the following:

F1) the DNA molecule whose coding sequence is SEQ ID No.2;

F2) the DNA molecule whose nucleotide sequence is SEQ ID No.2;

F3) The coding sequence is the DNA molecule shown in positions 1 to 204 from the 5' end of SEQ ID No.2;

F4) The nucleotide sequence is the DNA molecule shown at positions 1 to 204 from the 5' end of SEQ ID No. 2.

Specifically, in one embodiment of the present invention, the increase in the expression level of the gene encoding the protein IbGER5 in the target plant is achieved by introducing the DNA molecule shown in SEQ ID No. 2 into the target plant.

Specifically, in one embodiment of the present invention, the reduction in the expression level of the gene encoding the protein IbGER5 in the target plant is performed by adding the DNA shown in SEQ ID No. 2 at positions 1 to 204 from the 5' end The molecule and the DNA molecule of the reverse complement sequence are introduced into the plant of interest.

In one embodiment of the present invention, the method for cultivating transgenic plants comprises the steps of:

(1) construct the recombinant vector that comprises the DNA molecule shown in SEQ ID NO.2;

(2) the recombinant vector constructed in step (1) is introduced into the plant of interest (such as crop or sweet potato);

(3) The transgenic plant is obtained through screening and identification.

The introduction refers to introduction by recombinant means, including but not limited to Agrobacterium-mediated transformation, biolistic methods, electroporation, in planta techniques, and the like.

In one embodiment of the present invention, the method for cultivating transgenic plants (stress-resistant plants, such as drought-resistant plants and/or salt-tolerant plants) comprises the steps of:

(1) Construction of RNA interference vector pFGC5941-IbGER5 that inhibits the expression of the target gene IbGER5;

(2) introducing the RNA interference vector pFGC5941-IbGER5 constructed in step (1) into a target plant (such as a crop or sweet potato);

(3) After screening and identification, a stress-resistant plant with a stress resistance higher than that of the target plant is obtained.

In the above method, the plant can be any of the following:

G1) monocotyledonous or dicotyledonous plants;

G2) Convolvulus;

G3) sweet potato;

G4) sweet potato group plants;

G5) Sweet potato.

Specifically, the sweet potato can be a sweet potato variety, chestnut fragrant.

The protein IbGER5, and/or the biological material are also within the scope of the present invention.

Herein, the stress resistance may be salt tolerance and/or drought tolerance.

Herein, the plant may be a crop (eg, a crop).

The present invention also provides the application of the method for cultivating transgenic plants in creating plants with altered stress resistance, and/or in plant breeding or improvement of plant germplasm resources.

The plants with altered stress resistance may be plants with increased or decreased stress resistance, such as drought-resistant plants, salt-tolerant plants, etc., but are not limited thereto.

Modulating plant stress resistance as described herein can be up-regulating (increasing) or down-regulating (decreasing) plant stress resistance.

Further, the modulating plant stress resistance may be up-regulating (increasing) or down-regulating (decreasing) salt tolerance and/or drought resistance of sweet potato.

Herein, the transgenic plant is understood to include not only the first-generation transgenic plants obtained by transforming the IbGER5 gene into a target plant or knocking out the IbGER5 gene, but also its progeny. The gene can be propagated in the species, and conventional breeding techniques can be used to transfer the gene into other varieties of the same species, including in particular commercial varieties. The transgenic plants include seeds, callus, whole plants and cells.

In the present invention, the IbGER5 gene that regulates plant stress resistance derived from sweet potato (Ipomoea batatas (L.) Lam.) is introduced into the recipient plant sweet potato variety Lizixiang to obtain a transgenic sweet potato plant overexpressing the IbGER5 gene. The plants were identified for their salt tolerance and drought resistance, and the results of various physiological and biochemical indicators showed that compared with the non-transgenic control sweet potato variety Lizixiang (WT), the transgenic sweetpotato plants overexpressing the IbGER5 gene were under salt stress and/or drought stress conditions. The results of in vitro identification, hydroponic identification and soil cultivation identification significantly reduced the stress resistance of sweet potato, that is, the salt tolerance and drought resistance of the transgenic sweet potato plants overexpressing the IbGER5 gene were significantly reduced.

The present invention obtains a transgenic sweet potato plant that interferes with the expression of the IbGER5 gene by introducing the IbGER5 gene fragment that regulates plant stress resistance from sweet potato (Ipomoea batatas (L.) Lam.) into the recipient plant sweet potato variety Lizixiang. The transgenic plants were identified for salt tolerance and drought resistance, and the results of various physiological and biochemical indicators showed that compared with the non-transgenic control sweet potato variety Lizixiang (WT), the transgenic sweetpotato plants that interfered with the expression of the IbGER5 gene were susceptible to salt stress and/or drought stress. Under these conditions, the in vitro identification improved the stress resistance of sweet potato, that is, the salt tolerance and drought resistance of the transgenic sweet potato plants that interfered with the expression of IbGER5 gene were improved.

To sum up, the IbGER5 protein of the present invention and its encoding gene IbGER5 can regulate the stress resistance (such as salt tolerance and/or drought resistance) of plants, by reducing the content and/or activity of IbGER5 protein in the target plant (such as inhibiting IbGER5 gene expression) to breed salt- and/or drought-tolerant plants. The IbGER5 protein and its encoding gene provided by the present invention have important theoretical significance and application value in regulating the salt tolerance and drought resistance of sweet potato.

Description of drawings

Figure 1 shows the electrophoresis of PCR detection of transgenic plants. Wherein, lane M is the Maker band, lane W is the band of the negative control (water); lane P is the positive control (recombinant plasmid pCAMBIA1300-GFP-IbGER5 (A in Figure 1) or pFGC5941-IbGER5 (B in Figure 1) ) band; lane WT is the band of the control sweet potato chestnut fragrant plant; lane OE-G1, OE-G2, OE-G4, OE-G34, OE-G53 is the sweet potato pseudo-transgenic plant transformed with pCAMBIA1300-GFP-IbGER5 Bands; lanes Ri-G6 and Ri-G7 are the bands of the transgenic plants transformed with pFGC5941-IbGER5 sweet potato.

Figure 2 is a graph showing the results of identification of salt tolerance and drought resistance of transgenic plants under in vitro culture.

Figure 3 is a graph showing the results of identification of salt tolerance and drought resistance of transgenic plants under hydroponic conditions.

Figure 4 is a graph showing the results of identification of drought resistance of transgenic plants under soil cultivation conditions.

Fig. 5 is a graph showing the measurement results of physiological and biochemical indexes of transgenic plants after salt stress and drought stress treatment.

Detailed ways

The present invention will be further described in detail below with reference to the specific embodiments, and the given examples are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can serve as a guide for those of ordinary skill in the art to make further improvements, and are not intended to limit the present invention in any way.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product specification. The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

The sweet potato lines Lizixiang and ND98 in the following examples are described in the following documents: Zhang Huan. Analysis of the salt-tolerant transcriptome of sweet potato and cloning and functional verification of the stress resistance-related genes IbBBX24 and IbCPK28. Doctoral dissertation of China Agricultural University, 2017. The public is available from the Laboratory of Sweetpotato Genetics and Breeding of China Agricultural University to replicate this experiment.

The pMD19-T vector in the following examples is the product of Bao Bioengineering (Dalian) Company, and the product catalog number is 6013. The pCAMBIA1300 vector is a product of Shanghai Lianmai Bioengineering Co., Ltd., and the product catalog number is LM1375. The pFGC5941 vector is a product of Beijing Huayueyang Biotechnology Co., Ltd., and the product catalog number is VECT0360.

In the following examples, EcoRI enzyme is a product of Thermo Fisher Scientific (China) Co., Ltd., and the product catalog number is FD0274. SacI enzyme is a product of Thermo Fisher Scientific (China) Co., Ltd., and the product catalog number is FD1133. KpnI enzyme is a product of Thermo Fisher Scientific (China) Co., Ltd., and the catalog number is FD0524. SalI enzyme is a product of Thermo Fisher Scientific (China) Co., Ltd., and the catalog number is FD0644. PstI enzyme is a product of Thermo Fisher Scientific (China) Co., Ltd., and the catalog number is FD0614. XhoI enzyme is a product of Thermo Fisher Scientific (China) Co., Ltd., and the catalog number is FD0694. SwaI enzyme is a product of Thermo Fisher Scientific (China) Co., Ltd., and the product catalog number is FD1244. BamHI enzyme is a product of Thermo Fisher Scientific (China) Co., Ltd., and the catalog number is FD0054. XbaI enzyme is a product of Thermo Fisher Scientific (China) Co., Ltd., and the catalog number is FD0684. Escherichia coli DH5α is a product of Shenzhen Kangti Life Technology Co., Ltd., the product catalog number is KTSM101L, and Agrobacterium tumefaciens EHA105 is a product of Beijing Qingke Biotechnology Co., Ltd., and the product catalog number is TSC-A03.

The following examples use SPSS statistical software to process the data, the experimental results are expressed as mean ± standard deviation, using T test, P < 0.05 (*) indicates a significant difference, P < 0.01 (**) indicates a very significant difference Sexual differences were analyzed by one-way ANOVA, with different letters indicating statistically significant differences.

The quantitative tests in the following examples, unless otherwise specified, were set up to repeat the experiments three times, and the results were averaged.

Example 1. Application of protein IbGER5 in regulating sweet potato stress resistance

The inventor of the present application isolated and cloned a sweet potato gene from the sweet potato line ND98, and named it IbGER5. The nucleotide sequence of the coding sequence (CDS) of the gene IbGER5 is shown in SEQ ID NO.2; the amino acid sequence encoded by the gene IbGER5 is the protein IbGER5 of SEQ ID NO.1, which consists of 283 amino acid residues.

1. Construction of plant expression vector

A. Construction of recombinant vector pCAMBIA1300-GFP-IbGER5

According to the coding sequence (SEQ ID NO.2) of sweet potato IbGER5 protein nucleotides, the primer sequences for amplifying the complete coding sequence were designed, and the forward and reverse primers were introduced into KpnI and SalI restriction sites respectively, and the primer sequences were as follows:

IbGER5-OE-KpnI:

5'-TACGAATTCGAGCTC GGTACC ATGGAGCCAAAAGGAGAAGAAT-3' (the underlined part is the KpnI restriction site),

IbGER5-OE-SalI:

5'-CTTGCATGCCTGCAG GTCGAC GTTAGCAGTAGCAGGTTGTGACA-3' (the underlined part is the SalI restriction site).

Using the double-stranded DNA molecule shown in SEQ ID NO.2 in the artificially synthesized sequence table as a template, with IbGER5-OE-KpnI and IbGER5-OE-SalI as primers for PCR amplification, the product was connected to the pMD19-T carrier The recombinant vector was obtained, named pMD-IbGER5, and the sequencing of M13-F/R (5'-GTAAAACGACGGCCAGT-3'/5'-CAGGAAACAGCTATGAC-3') was performed to ensure the reading frame and enzyme of sweet potato IbGER5 protein nucleotides. The cut point is correct.

The plant expression vector pCAMBIA1300 was digested with the restriction enzymes EcoRI and SacI, and a fragment of about 8948 bp was recovered. The primers (5'-CGAGCTCTCTCTATCTAAACATCTCTCTCTGACC-3' and 5'-CGGAATTCGCTGTGAGAGCAGATGAGGTTC-3') were cloned from the vector pCAMBIA1300 to carry EcoRI. The 770bp CaMV 35S fragment of the SacI restriction site was recovered, and the two fragments were ligated to obtain the recombinant plasmid pCAMBIA1300-1.

The recombinant plasmid pCAMBIA1300-1 was digested with restriction enzymes SalI and PstI, and a fragment of about 9708 bp was recovered. The primers (5'-GCGTCGACATGGTGAGCAAGGGCGAG-3' and 5'-AACTGCAGTTACTTGTACAGCTCGTCCATGC-3') were cloned to carry SalI and PstI enzymes. The 720bp GFP fragment at the cut site was recovered, and the two fragments were ligated to obtain the recombinant plasmid pCAMBIA1300-GFP.

The pCAMBIA1300-GFP recombinant vector was double digested with KpnI and SalI, and the large fragment of the vector was recovered. The recombinant vector pMD-IbGER5 was double digested with restriction enzymes KpnI and SalI, and a small DNA fragment of about 852 bp was recovered. The recovered vector was large. The fragment is ligated with a small DNA fragment to obtain a recombinant vector pCAMBIA1300-GFP-IbGER5, which is the target plasmid. The target plasmid was transformed into Escherichia coli DH5α, cultured at 37°C for 20h, PCR analysis and restriction enzyme digestion identification of the recombinant vector pCAMBIA1300-GFP-IbGER5 were performed, and sequencing was performed for verification. The sequencing results showed that the sequence shown in SEQ ID No. 2 in the sequence listing was inserted between the KpnI and SalI restriction sites of the vector pCAMBIA1300-GFP, indicating that the recombinant vector was constructed correctly.

The recombinant vector pCAMBIA1300-GFP-IbGER5 is to replace the fragment (small fragment) between the KpnI and SalI recognition sites of the pCAMBIA1300-GFP vector with a DNA fragment whose nucleotide sequence is SEQ ID No. 2 in the sequence table, keeping pCAMBIA1300-GFP The other sequences of the vector remain unchanged, and the obtained recombinant expression vector. The recombinant vector pCAMBIA1300-GFP-IbGER5 expresses the protein IbGER5 shown in SEQ ID No. 1 in the sequence listing.

The recombinant vector pCAMBIA1300-GFP-IbGER5 has an expression cassette, and the nucleotide sequence of the expression cassette includes the CaMV35S promoter, the gene encoding IbGER5 protein (SEQ ID No. 2) and the gene encoding green fluorescent protein (GFP).

B. Construction of recombinant vector pFGC5941-IbGER5

Taking the double-stranded DNA molecule shown in SEQ ID NO.2 in the artificially synthesized sequence table as a template, using IbGER5-Ri-UF(XhoI): 5'-TTTGGAGAGGACACG CTCGAG ATGGAGCCAAAAGGAGAAG AATCA-3' (underlined as restriction endonuclease) Recognition sequence of XhoI) and IbGER5-Ri-UR(SwaI): 5'-AAGAAATTCTTACAC ATTTAAAT AGGTTGAGGGTGGAAATCTTGG-3' (underlined is the recognition sequence of restriction endonuclease SwaI) after PCR amplification as primers, a small molecule of about 200bp was recovered. The plant expression vector pFGC5941 was double digested with restriction enzymes XhoI and SwaI, and a large vector fragment of about 10 kb was recovered. The recovered large vector fragment was ligated with the small DNA fragment to obtain the recombinant vector pFGC5941-IbGER5-U1.

Take the double-stranded DNA molecule shown in SEQ ID NO.2 in the synthetic sequence table as the template, take IbGER5-Ri-DF(BamHI): 5'-AATTTGCAGGTATTT GGATCC AGGTTGAGGGTGGAAATCTTGG-3' (the underline is the restriction endonuclease BamHI) Recognition sequence of XbaI) and IbGER5-Ri-DR(XbaI): 5'-GGTCTTAATTAACTC TCTAGA ATGGAGCCAAAAGGAGAAGAATCA-3' (underlined is the recognition sequence of restriction endonuclease XbaI) after PCR amplification as primers, a small fragment of about 200bp is recovered. , the plant expression vector pFGC5941-IbGER5-U1 was double digested with restriction endonucleases BamHI and XbaI, the large vector fragment of about 10 kb was recovered, and the recovered vector large fragment was connected with the small DNA fragment to obtain the recombinant vector pFGC5941-IbGER5, namely target plasmid. The sequencing results showed that the DNA molecules shown in the sequence shown in SEQ ID No. 2 in the sequence table from the 1st to 204th positions from the 5' end were inserted between the XhoI and SwaI restriction sites of the vector pFGC5941, BamHI and XbaI enzymes Between the cleavage sites, the reverse complementary sequence of the DNA molecule shown at positions 1 to 204 from the 5' end of the sequence shown in SEQ ID No. 2 in the Sequence Listing is inserted.

The recombinant vector pFGC5941-IbGER5 is to replace the small fragment between the XhoI and SwaI recognition sequences of the pFGC5941 vector with the DNA molecule fragment shown in the 1st to 204th position from the 5' end of SEQ ID No.2 in the sequence listing. The fragment (small fragment) between the recognition sites of Dicer BamHI and XbaI is replaced with the nucleotide sequence that is the reverse complement of the DNA molecule shown in the 5' end of SEQ ID No. 2 in the sequence listing. , keeping the other sequences of the pFGC5941 vector unchanged, and the obtained recombinant expression vector. The recombinant expression vector pFGC5941-IbGER5 interferes with the expression of the protein IbGER5 shown in SEQ ID No. 1 in the sequence listing.

The recombinant vector pFGC5941-IbGER5 has an expression cassette, and the nucleotide sequence of the expression cassette contains the CaMV35S promoter, the gene fragment encoding the IbGER5 protein and the OCS polyA terminator.

2. Plant expression vector transformed into Agrobacterium

(1) Thaw the prepared Agrobacterium EHA105 competent cells on ice, add 2 μg of the extracted pCAMBI A1300-GFP-IbGER5 or pFGC5941-IbGER5 plasmids, flick the tube wall to mix, and take an ice bath for 10 minutes;

(2) Quick-freeze in liquid nitrogen for 5 minutes, water bath at 37°C for 10 minutes, and ice bath for 5 minutes;

(3) Add 600 μL of liquid LB medium, culture at 28°C and 200rpm for 5h;

(4) Spread 200 μL of bacterial liquid on LB solid medium containing 100ug/mL kanamycin and 100ug/mL rifampicin;

(5) Invert the dark culture at 28°C for 2 days, take an appropriate amount of Agrobacterium and cultivate it in liquid LB medium for later use, to obtain the Agrobacterium liquid introduced into the pCAMBIA1300-GFP-IbGER5 or pFGC5941-IbGER5 vector, and the recombinant Agrobacterium is named as EHA105/ pCAMBIA1300-GFP-IbGER5 or EHA105/pFGC5941-IbGER5.

3. Genetic transformation and regeneration of sweet potato

A. Transformation and regeneration of sweet potato overexpressed transgene-positive plants

EHA105/pCAMBIA1300-GFP-IbGER5 was introduced into sweet potato cultivar Lizixiang by Agrobacterium-mediated method. The specific method is as follows:

(1) Peel the shoot tip meristem of sweet potato variety Lizixiang, place it on MS solid medium containing 2.0 mg/L 2,4-D, and cultivate at 27±1°C for 8 weeks to obtain embryogenic callus;

(2) Put the embryogenic callus into MS liquid medium containing 2.0 mg/L 2,4-D, and place it on a shaker for horizontal shaking culture for 8 weeks to obtain embryogenic cell clusters with a diameter of 0.7-1.3 mm. ;

(3) The embryogenic cell mass is screened through a 20-mesh mesh screen, and the larger cell mass is transferred to a 30-mesh mesh screen, and lightly ground to make a wound in the embryogenic cell mass, and the larger embryogenic cell mass after grinding is oscillated. cultured for 3 days;

(4) EHA105/pCAMBIA1300-GFP-IbGER5 was transformed into embryogenic cell mass by Agrobacterium-mediated method, and then placed in co-culture medium (MS solid culture medium containing 30 mg/L AS, 2.0 mg/L 2,4-D) base), cultivated in the dark at 28°C for 3 days;

(5) The embryogenic cell mass was washed once in MS liquid medium containing 400 mg/L cefotaxime sodium (CS) and 2.0 mg/L 2,4-D, and then washed once in MS liquid medium containing 2.0 mg/L 2, Shake culture in 4-D MS liquid medium for 1 week;

(6) Place the embryogenic cell mass on the screening medium (MS solid medium containing 100 mg/L CS, 5 mg/L hygromycin (Hyg), 2,4-D), and culture at 28°C in the dark for 10-12 weeks, in which the medium is changed every two weeks;

(7) Place the embryogenic cell mass on the somatic embryo induction medium (MS solid medium containing 100 mg/L CS and 1.0 mg/L ABA), and culture it alternately between light and dark at 28°C for 2-4 weeks to acquire resistance callus;

(8) Place the resistant callus on MS solid medium, and culture it alternately between light and dark at 28°C for 4-8 weeks, to obtain 5 overexpressing transgenic plants to be identified (that is, to be overexpressed transgenic plants), and name them respectively. For OE-G1, OE-G2, OE-G4, OE-G34, OE-G53.

(9) Extract the genomic DNA of the leaves of the overexpressed transgenic plants (OE-G1, OE-G2, OE-G4, OE-G34, OE-G53) by CTAB method, take the extracted genomic DNA as a template, water and control chestnuts The fragrant plant is the negative control, the plasmid pCAMBIA1300-GFP-IbGER5 is the positive control, and 35S-F and IbGER5-R are used as primers for PCR amplification to obtain the PCR amplification product; if the PCR amplification product contains a band of about 1090bp, The corresponding sweet potato transgenic plants to be identified are the sweet potato overexpression transgenic positive plants. The primers 35S-F and IbGER5-R sequences are shown below:

35S-F: 5'-TGACGCACAATCCCACTATCCT-3'

IbGER5-R: 5'-GTTAGCAGTAGCAGGTTGTGACA-3'

The results of electrophoresis detection and amplification are shown in A in Figure 1 (A in Figure 1, lane M is shown as the Maker band, and lane W is shown as the band of the negative control (water); lane P is shown as the positive control (recombinant plasmid pCAMBIA1300-GFP- IbGER5); lane WT is shown as the band of sweet potato lichen fragrant plant; lanes OE-G1, OE-G2, OE-G4, OE-G34, OE-G53 are shown as sweet potato clones transformed with pCAMBIA1300-GFP-IbGER5 The bands of expressing transgenic plants can be seen from A in Figure 1. Lanes OE-G1, OE-G2, OE-G4, OE-G34, OE-G53 and the positive control all amplified a target band of 852 bp, indicating that the IbGER5 gene It has been integrated into the genome of sweet potato chestnut incense, and it is proved that these regenerated plants are overexpressed transgenic plants. The sweet potato plants identified as overexpressing transgenes are propagated by asexual propagation (plants obtained by propagating a transgenic seedling are used as One strain), the resistance identification of salt tolerance and drought resistance was carried out.

B. Transformation and regeneration of sweet potato RNAi-positive plants

EHA105/pFGC5941-IbGER5 was introduced into sweet potato cultivar Lizixiang by Agrobacterium-mediated method.

(1) According to the method of steps (1) to (8) in A, replace EHA105/pCAMBIA1300-GFP-IbGER5 with EHA105/pFGC5941-IbGER5, and replace the screening medium with 100mg/L CS, 0.3mg/L herbicide (PPT), 2,4-D MS solid medium, other steps were unchanged, and 2 sweet potato interfering transgenic plants to be identified (ie pseudo-RNAi plants) were obtained, which were named Ri-G6 and Ri-G7 in turn.

(2) Extract genomic DNA from leaves of pseudo-RNAi plants (Ri-G6, Ri-G7), use the extracted genomic DNA as a template, water and control chestnut incense plants as a negative control, plasmid pFGC5941-IbGER5 as a positive control, with int-F (5'-CAACCACAAAAGTATCTATGAGCCT-3') and int-R (5'-TTCACATGTCAGAAACATTCTGATG-3') are primers for PCR amplification to obtain a PCR amplification product; if the PCR amplification product contains a band of about 888bp, the corresponding Sweet potato transgenic plants to be identified are sweet potato RNAi-positive plants.

The results of electrophoresis detection and amplification are shown in B in Figure 1 (B in Figure 1, lane M is shown as the Maker band, and lane W is shown as the band of the negative control (water); lane P is shown as the positive control (recombinant plasmid pFGC5941-IbGER5) Swimming lane WT shows the band of sweet potato and chestnut fragrant plant; lane Ri-G6, Ri-G7 shows the band of sweet potato pseudo-RNAi plant transformed with pFGC5941-IbGER5, as can be seen from B in Figure 1, lane Ri-G6 , Ri-G7 and positive control all amplified the target band of 888bp, indicating that the pFGC5941-IbGER5 vector has been transferred into the genome of sweet potato chestnut, and proved that these regenerated plants are RNAi plants. The sweet potato plants identified as RNAi The method of vegetative propagation was used for propagation (a plant obtained by propagation of a transgenic seedling was regarded as a line), and the resistance identification of salt tolerance and drought resistance was carried out.

4. Salt tolerance identification of transgenic plants

Tested plants: sweet potato variety Lizixiang as a control plant (WT); plants with line numbers OE-G1, OE-G2, OE-G4, OE-G34, OE-G53, Ri-G6, and Ri-G7, respectively, 3 plants per line.

The IbGER5 transgenic lines (line numbers: OE-G1, OE-G2, OE-G4, OE-G34, OE-G53, Ri-G6, Ri-G7) and chestnut incense control plants (WT) were in normal After 4 weeks of stress culture on MS medium and MS medium containing 150 mM NaCl, the growth status of the plants was observed, and the root lengths of the plants were measured. The results are shown in A, B, D and E in Figure 2 (A in Figure 2 is the growth status of sweet potato plants under normal growth conditions, B in Figure 2 is the growth status of sweet potato plants under salt stress, D in Figure 2 is the statistical result of the root length of sweet potato plants under normal growth conditions, and E in Figure 2 is the root of sweet potato plants under salt stress long statistics). It can be found that the root length of the overexpressed transgenic plants (OE-G1, OE-G2, OE-G4, OE-G34, OE-G53) was significantly lower than that of the control chestnut incense (WT) after salt stress treatment (150 mM NaCl). The root length of RNAi plants (Ri-G6, Ri-G7) was higher than that of the control chestnut incense (WT). The results of in vitro identification showed that the salt tolerance of the overexpressed transgenic plants was significantly reduced, and the salt tolerance of the RNAi plants was improved. The IbGER5 gene Or the protein IbGER5 can regulate the salt tolerance of plants.

To further verify whether overexpression of IbGER5 gene can improve the salt tolerance of transgenic sweet potato under hydroponic conditions. Plants of transgenic lines (line numbers OE-G1, OE-G2, OE-G4, OE-G34, OE-G53) overexpressing the IbGER5 gene and stems of control chestnut incense plants (WT) were retrieved from the isolated field Each section was cut into 25 cm, and one stem node was placed in normal Hoagland solution and Hoagland solution containing 150 mM NaCl respectively. The stress was treated for 4 weeks, and the medium was changed every 7 days to observe the growth status of the plant. , the results of measuring the root length of the plant are shown in A, B, D and E in Figure 3 (the left picture in A and B in Figure 3 is the growth condition of sweet potato plants in Hoagland solution, the right picture is taken out from the solution after washing In Figure 3, A is the growth status of sweetpotato plants under normal growth conditions, B in Figure 3 is the growth status of sweetpotato plants under salt stress, and D in Figure 3 is the statistical result of root length of sweetpotato plants under normal growth conditions, and in Figure 3 E is the statistical result of root length of sweet potato plants under salt stress). It can be found that after salt stress treatment (150mM NaCl), the root length of the overexpressed transgenic plants was significantly shorter than that of the control chestnut incense (WT). The results of hydroponic identification showed that the salt tolerance of the overexpressed transgenic plants was significantly reduced. Significantly reduce the salt tolerance of plants, the IbGER5 gene or protein IbGER5 has the function of regulating the salt tolerance of plants.

5. Identification of drought resistance of transgenic plants

Plants of IbGER5 transgenic lines (line numbers: OE-G1, OE-G2, OE-G4, OE-G34, OE-G53, Ri-G6, Ri-G7) and control chestnut incense plants (WT) After 4 weeks of stress culture on the MS medium of 20% PEG6000 simulating drought stress, the growth status of the plant was observed, and the root length of the plant was measured. The results are shown in C in Figure 2 and F in Figure 2 (C in Figure 2 is The growth status of sweet potato plants under drought stress, F in Figure 2 is the statistical result of root length of sweet potato plants under drought stress). It can be found that after drought stress treatment (20% PEG6000), the root length of the overexpressed transgenic plants was significantly lower than that of the control Lizixiang (WT), and the growth status was significantly worse than that of the control plants, while the RNAi plants (Ri-G6, Ri-G7 ) root length was higher than that of the control chestnut incense (WT), and the growth state was good. The results of in vitro identification showed that the drought resistance of the overexpressed transgenic plants was significantly reduced, and the drought resistance of the RNAi plants was improved, and the overexpression of the IbGER5 gene could significantly reduce the drought resistance of the plants. , Interfering with the expression of IbGER5 gene can improve the drought resistance of plants, and IbGER5 gene or protein IbGER5 can regulate the drought resistance of plants.

To further verify whether overexpression of IbGER5 gene can improve the drought resistance of transgenic sweet potato under hydroponic conditions. Plants of transgenic lines (line numbers OE-G1, OE-G2, OE-G4, OE-G34, OE-G53) overexpressing the IbGER5 gene and stems of control chestnut incense plants (WT) were retrieved from the isolated field Each segment was cut into 25cm, and one stem node was placed in Hoagland solution containing 20% PEG6000, simulated drought stress for 2 weeks, then rehydrated for 2 weeks, and the medium was changed every 7 days to observe the growth of the plant. , measure the root length of the plant, the results are shown in C in Figure 3 and F in Figure 3 (the left picture in C in Figure 3 is the growth condition of the sweet potato plant under drought stress, and the right picture is the sweet potato plant after taking out the cleaning from the solution. , F in Figure 3 is the statistical result of root length of sweet potato plants under drought stress). It can be found that after drought stress treatment (20% PEG6000), the root length of the transgenic plants is significantly shorter than that of the control chestnut incense (WT), and the growth state is significantly worse than that of the control plants, showing more severe wilting than the control. It shows that the drought resistance of transgenic plants is significantly reduced, and the overexpression of IbGER5 gene can significantly reduce the drought resistance of plants. IbGER5 gene or protein IbGER5 has the function of regulating the drought resistance of plants.

To further verify whether overexpression of IbGER5 gene can improve the drought resistance of transgenic sweet potato under soil cultivation conditions. Plants of transgenic lines (line numbers: OE-G1, OE-G2, OE-G4) overexpressing the IbGER5 gene and the stem segments of the control chestnut incense plant (WT) were retrieved from the isolated field, and each segment was cut into 20 cm , to ensure that a stem node is inserted into the soil, water for 2 weeks until the stem segment grows normally, and then carry out drought treatment (that is, do not water for 8 consecutive weeks), observe the growth state of the plant after 8 weeks, measure the root length and number of roots of the plant, and the results are shown in Figure 4 (A in Figure 4 is the growth status of sweet potato plants in dry ponds, B in Figure 4 is the sweet potato plant taken out from the soil, and C in Figure 4 is the statistical sweet potato plant phenotype index). It can be found that after drought stress treatment, the root length of the transgenic plants was significantly shorter than that of the control chestnut incense (WT), the number of roots was less, and the growth state was significantly worse than that of the control plants, showing more severe wilting than the control. The dry pond results showed that The drought resistance of transgenic plants was significantly reduced. Overexpression of IbGER5 gene can significantly reduce the drought resistance of plants. IbGER5 gene or protein IbGER5 has the function of regulating the drought resistance of plants.

6. Determination of physiological and biochemical indicators

Tested plants: sweet potato variety Lizixiang as control plant (WT); plants with line numbers OE-G1, OE-G2, OE-G4, Ri-G6, Ri-G7, with 3 plants per line.

(1) Determination of H ₂ O ₂ content

H ₂ O ₂ is the most common reactive oxygen species in plants. It can directly or indirectly oxidize biological macromolecules such as nucleic acids and proteins in cells, damage the cell membrane, and accelerate the aging and disintegration of cells. Therefore, the higher the _{H2O2 content} , the greater the degree of damage to the plant by the stress.

References (Zhang H, Gao X, Zhi Y, et al. A non-tandem CCCH-type zinc-fingerprotein, IbC3H18, functions as a nuclear transcriptional activator and enhancesabiotic stress tolerance in sweet potato [J]. New Phytologist, 2019, 223:1918-1936) DAB and NBT staining methods, DAB and NBT staining were performed on sweet potato leaves, respectively. The hydrogen peroxide content (H ₂ O ₂ ) kit of Suzhou Keming Biotechnology Co., Ltd. was used to detect the H ₂ O ₂ content in sweet potato plants. The dyed sweet potato leaves were treated with salt tolerance and drought resistance for 10 days in vitro, and the plants for content determination were the plants treated with salt tolerance and drought resistance for 4 weeks in vitro. The experiment was repeated 3 times, and the results were averaged.

The results are shown in A, B, C, D and E in Fig. 5 (A in Fig. 5 is the DAB staining result, and B in Fig. 5 is the relative intensity of DAB staining spots calculated by imageJ software, with the control chestnut incense in normal MS medium The spot intensity of the plant is taken as 100%, C in Figure 5 is the result of NBT staining, D in Figure 5 is the relative intensity of the NBT stained spots calculated by imageJ software, and the spot intensity of the control chestnut incense plant in normal MS medium is taken as 100% , E in Figure 5 is the statistical result of H ₂ O ₂ content). It can be found that the H ₂ O ₂ content of the transgenic plants (line numbers OE-G1, OE-G2, OE-G4) overexpressing the IbGER5 gene after the salt stress treatment of 150 mM NaCl and the simulated drought stress treatment of 20% PEG6000 It was significantly higher than that of the control chestnut incense plants, while the H ₂ O ₂ content of the RNAi plants (strain numbers Ri-G6, Ri-G7) was significantly lower than that of the control chestnut incense plants.

(2) Determination of malondialdehyde (MDA) content

When plant organs age or suffer damage under adversity, oxygen free radicals often act on unsaturated fatty acids of lipids to generate lipid peroxidation, and MDA is the final decomposition product of lipid peroxidation, and its content can reflect the damage of plants to adversity Degree. Therefore, the higher the MDA content, the greater the degree to which the plant suffered from the stress Shanghai.

The malondialdehyde (MDA) content kit of Suzhou Keming Biotechnology Co., Ltd. was used to detect the content of MDA in sweet potato plants. Sweet potato plants were treated for 4 weeks after in vitro identification of salt tolerance and drought resistance. The experiment was repeated 3 times, and the results were averaged.

The results are shown in Figure 5, F. It can be found that the MDA content of the transgenic plants overexpressing the IbGER5 gene (line numbers OE-G1, OE-G2, OE-G4) was significantly higher than that of the 150 mM NaCl salt stress treatment and 20% PEG6000 simulated drought stress treatment. Control chestnut incense plants, and RNAi plants (strain numbers Ri-G6, Ri-G7) MDA content was significantly lower than the control chestnut incense plants.

(3) Determination of proline (Pro) content

Proline is widely present in plants, and under adverse conditions, the proline content in plants increases significantly, and the increase reflects stress resistance to a certain extent. Therefore, proline can be used as a biochemical indicator of plant stress resistance.

The proline content in sweet potato plants was detected by the proline (PRO) content assay kit of Suzhou Keming Biotechnology Co., Ltd. Sweet potato plants were treated for 4 weeks after in vitro identification of salt tolerance and drought resistance. The experiment was repeated 3 times, and the results were averaged.

The results are shown in G in Figure 5. It can be found that the proline content of the transgenic plants overexpressing the IbGER5 gene (strain numbers OE-G1, OE-G2, OE-G4) was significantly higher after the salt stress treatment of 150 mM NaCl and the simulated drought stress treatment of 20% PEG6000. It was lower than that of the control chestnut incense plants, while the proline content of the RNAi plants (strain numbers Ri-G6 and Ri-G7) was significantly higher than that of the control chestnut incense plants.

(4) Peroxidase (POD) activity assay

POD (EC 1.11.1.7) is widely present in plants and can be used as a biochemical indicator of plant stress resistance. The lower the activity of POD, the greater the degree of damage to the plant.

The peroxidase (POD) kit of Suzhou Keming Biotechnology Co., Ltd. was used to detect the POD content in sweet potato plants. Sweet potato plants were treated for 4 weeks after in vitro identification of salt tolerance and drought resistance. The experiment was repeated 3 times, and the results were averaged.

The results are shown in Figure 5, H. It can be found that the POD activity of the transgenic plants overexpressing the IbGER5 gene (line numbers OE-G1, OE-G2, OE-G4) was significantly lower than that of the control after 150 mM NaCl salt stress treatment and 20% PEG6000 simulated drought stress treatment. Chestnut incense plants, and RNAi plants (strain numbers Ri-G6, Ri-G7) showed significantly higher POD activity than control chestnut incense plants.

In conclusion, under the conditions of 150 mM NaCl salt stress treatment and 20% PEG6000 simulated drought stress, overexpression of IbGER5 gene significantly increased H ₂ O ₂ and MDA content, decreased proline (Pro) content and POD activity. The activity of oxidases decreased, which increased the oxidative damage caused by salt stress and drought stress to transgenic plants, and greatly reduced the tolerance of overexpressed transgenic plants to stress, while interference with IbGER5 gene expression significantly reduced H ₂ O ₂ and MDA content, increased proline (Pro) content and POD activity, and increased the activity of antioxidant enzymes, thereby reducing the oxidative damage caused by salt stress and drought stress to transgenic plants, and greatly improving the resistance of RNAi plants to adversity stress. tolerance.

The above results showed that overexpression of IbGER5 gene in sweetpotato can significantly reduce the salt tolerance and drought resistance of sweetpotato plants, and interference with the expression of IbGER5 gene can significantly improve the salt tolerance and drought resistance of sweetpotato plants. The IbGER5 protein and its encoding gene IbGER5 of the present invention can regulate the stress resistance (such as salt tolerance and/or drought resistance) of plants, and can improve the content and/or activity of the IbGER5 protein in the target plant (such as overexpressing the IbGER5 gene) The stress resistance of the target plant is significantly reduced, and the stress resistance of the target plant can be significantly improved by reducing the content and/or activity of the IbGER5 protein in the target plant (eg, by interfering with the expression of the IbGER5 gene).

Example 2. The protein IbGER5 regulating sweet potato stress resistance and the acquisition of its encoding gene

Experimental material: The sweet potato strain ND98 was used as the experimental material.

1. Extraction of total RNA from sweet potato: Take 1 g of young leaves of sweet potato strain ND98 and grind it into powder in liquid nitrogen, add it to a 2 mL centrifuge tube, and extract total RNA from sweet potato by TransZol method, using PrimeScript ^TM RT reagent Kit with gDNA Eraser reagent Box (product of Kangwei Century Biotechnology (Beijing) Co., Ltd.) reverse-transcribed the first-strand cDNA.

2. In the published transcriptome of salt tolerance of sweet potato, obtain the EST sequence shown in SEQ ID No.3 in the sequence table, and compare it by searching in the Sweetpotato Garden library to obtain the EST sequence shown in SEQ ID No.4 in the sequence table. homologous sequence. According to the nucleotide sequence (SEQ ID No. 4) obtained by homology alignment, primers IbGER5-F and IbGER5-R were designed and synthesized, and the sequences are:

IbGER5-F: 5'-ATGGAGCCAAAAGGAGAAGAAT-3'

IbGER5-R: 5'-GTTAGCAGTAGCAGGTTGTGACA-3'

3. Using the cDNA obtained in step 1 as a template, and using the IbGER5-F and IbGER5-R synthesized in step 2 as primers, carry out PCR amplification to obtain a PCR amplified fragment product of about 852 bp and sequence it.

The results show that the nucleotide sequence of the PCR amplification product obtained in step 3 is shown in SEQ ID No. 2 in the sequence listing, the gene shown in this sequence is named IbGER5 gene, and the protein encoded by it is named IbGER5 protein or protein. IbGER5, the amino acid sequence is shown in SEQ ID No. 1 in the sequence listing.

The present invention has been described in detail above. For those skilled in the art, without departing from the spirit and scope of the present invention, and without unnecessary experimentation, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. While the invention has been given particular embodiments, it should be understood that the invention can be further modified. In conclusion, in accordance with the principles of the present invention, this application is intended to cover any alterations, uses or improvements of the invention, including changes made using conventional techniques known in the art, departing from the scope disclosed in this application. The application of some of the essential features can be made within the scope of the following appended claims.

SEQUENCE LISTING

<110> China Agricultural University

<120> Protein IbGER5 regulating plant stress resistance and its encoding gene and application

<160> 4

<170> PatentIn version 3.5

<210> 1

<211> 283

<212> PRT

<213> Sweet potato (Ipomoea batatas (L.) Lam.)

<400> 1

Met Glu Pro Lys Gly Glu Glu Ser Glu Pro His Lys Pro Leu Ser Ser

1 5 10 15

Ser Ser Ser Glu Ser Gln Ala Pro Ala Glu Met Asp Pro Gln Lys Trp

20 25 30

Gly Thr His Val Met Gly Arg Pro Ala Val Pro Thr Thr His Pro Asp

35 40 45

Asn Gln Lys Ala Ala Leu Trp Arg Ser Glu Asp Gln His Gln Asp Phe

50 55 60

His Pro Gln Pro Tyr Val Val Tyr Ser Pro Val Asp Arg Pro Pro Ser

65 70 75 80

Asn Asn Pro Phe Glu Ser Val Cys His Met Phe Asn Ser Trp Ser His

85 90 95

Arg Ala Glu Thr Val Ala Arg Asn Val Trp His Asn Leu Lys Thr Ala

100 105 110

Pro Ser Val Ser Glu Ala Ala Trp Gly Lys Leu Asn Met Thr Ala Lys

115 120 125

Ala Ile Thr Glu Gly Gly Phe Glu Ala Phe Tyr Lys Gln Ile Phe Ala

130 135 140

Thr Asp Pro Tyr Glu Lys Leu Lys Lys Thr Tyr Ala Cys Tyr Leu Ser

145 150 155 160

Thr Thr Thr Gly Pro Val Ala Gly Thr Leu Tyr Leu Ser Thr Thr Lys

165 170 175

Val Ala Phe Cys Ser Asp Arg Pro Leu Thr Phe Thr Ala Pro Ser Gly

180 185 190

Gln Glu Ala Trp Ser Tyr Tyr Lys Ile Ala Val Pro Leu Ala Asn Ile

195 200 205

Ala Ala Val Asn Pro Val Val Met Arg Glu Asn Pro Gln Glu Lys Tyr

210 215 220

Ile Gln Leu Val Thr Val Asp Gly His Asp Phe Trp Phe Met Gly Phe

225 230 235 240

Val Asn Phe Glu Lys Ala Thr His Asn Leu Leu Asp Gly Leu Ser Ser

245 250 255

Phe Arg Ala Tyr Gly Asn Asn Asn Ala Ala Gly Gln Ser Val Ser Gly

260 265 270

His Ala Asn Val Ser Gln Pro Ala Thr Ala Asn

275 280

<210> 2

<211> 852

<212> DNA

<213> Sweet potato (Ipomoea batatas (L.) Lam.)

<400> 2

atggagccaa aaggagaaga atcagagccc cataaaccct tgtcatcatc ttcttctgaa 60

tcccaagcac ctgcagaaat ggaccctcag aaatggggaa ctcatgtgat gggtcgtcct 120

gcagtcccca ccacccaccc ggataaccag aaggcggcct tgtggaggtc tgaagatcag 180

caccaagatt tccaccctca accttacgtc gtctattctc ccgtggatcg gccccccagc 240

aacaacccct ttgaatcagt gtgccacatg ttcaattctt ggagccatag agctgagact 300

gtagctcgta acgtatggca caacttgaaa actgcgccat ctgtatcaga agctgcttgg 360

ggaaagctga acatgactgc caaggcaata acagaaggtg gatttgaggc attttacaag 420

caaatctttg caaccgatcc ttatgagaag ctaaagaaga cgtatgcttg ttacctttca 480

acaacaaccg gccctgttgc cgggaccctc tatttgtcaa ccaccaaggt tgctttctgc 540

agtgatcgcc ctttgacctt cacagctcct tctggccagg aggcttggag ctactacaag 600

attgcagtac ctttggcaaa tatagcagca gtgaatccgg tggtaatgag agagaatcca 660

caagagaagt acattcagtt agtgacagtt gatggtcatg acttctggtt catggggttt 720

gtgaattttg agaaagcaac ccataatctc ctagacggct tgtcaagttt tagagcatat 780

ggaaataaca atgcagcagg gcagtctgtt agtggacatg cgaatgtgtc acaacctgct 840

actgctaact ag 852

<210> 3

<211> 1736

<212> DNA

<213> Sweet potato (Ipomoea batatas (L.) Lam.)

<400> 3

taattaataa ttaaaattga tatatgtatc ttggataaat acgaagtgga tggccggaaa 60

acccacacct ggaaacttcg ctatcatcta ttcaacggct ccacttgttg acacataaat 120

taagaaacaa ttactagcaa taataaatta gtaatataac gacgaacctt gttacttcta 180

cgcgtaatct cacgcttgtt ctagaacgat gctaaggttt ctattctgat cttaccagat 240

ttcttttccg gcccaattaa ccttcatctc aaagaaacca tcttcaatta atcctataaa 300

ttgcagtgtt ccgatcccca tctctatcta tctcaccact tctttaccaa agtatattct 360

tgccttcatt ccaattcaga aagatattcc cataagtccc caaataatgg agccaaaagg 420

agaagaatca gagccccata aacccttgtc atcatcttct tctgaatccc aagcacctgc 480

agaaatggac cctcagaaat ggggaactca tgtgatgggt cgtcctgcag tccccaccac 540

ccacccagat aaccagaagg ctgccttgtg gaggtctgag gatcattacc aagagttcca 600

ccctcaacct tacgtcgtct attctcccgt ggatcggccc cccagcaaca acccctttga 660

atcagtgtgc cacatgttca attcttggag ccatagagct gagactgtag ctcgtaacgt 720

atggcacaac ttgaaaactg cgccatctgt atcagaagct gcttggggaa agctgaacat 780

gactgccaag gcaataacag aaggtggatt tgaggcatat tacaagcaaa tctttgcaac 840

cgatccttat gagaagctaa agaagacgta tgcttgttac ctttcaacaa caaccggccc 900

tgttgccggg accctctatt tgtcaaccac caaggttgct ttctgcagtg atcgcccttt 960

gaccttcaca gctccttctg gccaggaggc ttggagctac tacaagattg cagtaccttt 1020

ggcaaatata gcagcagtga atccggtggt aatgagagag aatccacaag agaagtacat 1080

tcagttagtg acagttgatg gtcatgactt ctggttcatg gggtttgtga attttgagaa 1140

agcaacccat aatctcctag acggcttgtc aataaatttt agagcatatg gaaataataa 1200

tgcagcaggg cagtctgtga gtggacatgg gaatgtgtca caacttgcta gtgctaacta 1260

gcaataaagt agtatttcac cattcaactc ctcttacttg agttgtcttt ctgccatata 1320

cacagttatt gtattacttt gtgtgcttaa ttgcttggag tgtatccatg tatattgtta 1380

gttgttgttg ttggatgcat tatagatttt tcccttctgg gtatcgagga tgaggttgta 1440

ggcaatctgt gtagagaatt ttcaatataa actaaggggg tttcgtacat attctttagc 1500

ttaataatta cattgtttaa ttcgaagaaa ttacaattcc acggaattgt aacttcttct 1560

ttttgttgta ttgaaattca tactttcata tgaattctaa acaatgaagc atggaaaaga 1620

aaaagagaag agaaagattt gtttatcaat gaagcatgga aaagaaaaag agaagagaaa 1680

gatttgttta tcaatgaagc atggaaaaga aaaagagaag agaaagattt gtttat 1736

<210> 4

<211> 918

<212> DNA

<213> Sweet potato (Ipomoea batatas (L.) Lam.)

<220>

<221> misc_feature

<222> (555)..(555)

<223> n is a, c, g, or t

<400> 4

atggagccaa aaggagaaga atcagagccc cataaaccct tgtcagaaat ggaccctcag 60

aaatgggggaa ctcatgtgat gggtcgtcct gcagtcccca ccacccaccc ggataaccag 120

aaggctgcct tgtggaggtc tgaggatcag caccaagatt tccaccctca accttacgtc 180

gtctattctc ccgtggatcg gccccccagc aacaacccct ttgaatcagt gtgccacatg 240

ttcaattctt ggagccatag agctgagacc gtagctcgta acgtatggca caactcacaa 300

gagtcaatat tgactccact aaggctcgaa accaccaccc ctcttgaagc acaagagtcg 360

gtattgtctt cactgaggct ccacctcccg tataaaggaa aggtgaaaac tgcgccatcc 420

gtatcagaag ctgcttgggg aaagctgaac atgactgcca aggcaataac agaaggtgga 480

tttgaggcat attacaagca aatctttgca accgatccta atgagaagct aaagaagacc 540

tccttctggc caggnggccc tgttgccggg accctctatt tgtcaaccac caaggttgct 600

ttctgcagtg atcgcccttt gaccttcaca gctccttctg gccaggaggc ttggagctac 660

tacaagattg cagtaccttt ggcaaatata gcagcagtga atccggtggt aatgagagag 720

aatccacaag agaagtacat tcagatagtg acagttgatg gtcatgactt ctggttcatg 780

gggtttgtga attttgataa agcaacccat aatctcctag acggcttgtc aaattttaga 840

gcatatggaa ataataatgc agcagggcat tctgtgagtg gacatgcgaa tgtgtcacaa 900

cctgctactg ctaactag 918

Claims (10)

1. the application of protein or the substance regulating the activity and/or content of the protein, characterized in that the application is any of the following: D1) the application of protein or substances regulating the activity and/or content of the protein in regulating plant stress resistance; D2) the application of protein or substances regulating the activity and/or content of the protein in the preparation of products for regulating plant stress resistance; D3) application of protein or substances regulating the activity and/or content of said protein in cultivating stress-resistant plants; D4) the application of protein or substances regulating the activity and/or content of the protein in the preparation of products for cultivating stress-resistant plants; D5) Application of protein or substances regulating the activity and/or content of said protein in plant breeding; The protein is any of the following: A1) the amino acid sequence is the protein of SEQ ID No. 1; A2) A protein with more than 80% identity and the same function as the protein shown in A1) obtained by substitution and/or deletion and/or addition of amino acid residues to the amino acid sequence shown in SEQ ID No.1; A3) A fusion protein with the same function obtained by linking a tag to the N-terminus and/or C-terminus of A1) or A2). 2. The application according to claim 1, wherein the protein is derived from sweet potato. 3. The application of the biological material related to the protein described in claim 1 or 2, wherein the application is any of the following: E1) the application of the biological material related to the protein described in claim 1 or 2 in regulating plant stress resistance; E2) application of the biological material related to the protein described in claim 1 or 2 in the preparation of a product for regulating plant stress resistance; E3) the application of the biological material related to the protein described in claim 1 or 2 in cultivating stress-resistant plants; E4) application of the biological material related to the protein described in claim 1 or 2 in the preparation of a product for cultivating stress-resistant plants; E5) the application of the biological material related to the protein described in claim 1 or 2 in plant breeding; The biological material is any one of the following B1) to B8): B1) a nucleic acid molecule encoding the protein of claim 1 or 2; B2) a nucleic acid molecule that inhibits or reduces the expression of a gene encoding the protein of claim 1 or 2; B3) an expression cassette containing the nucleic acid molecule of B1) and/or B2); B4) a recombinant vector containing the nucleic acid molecule described in B1) and/or B2), or a recombinant vector containing the expression cassette described in B3); B5) a recombinant microorganism containing the nucleic acid molecule described in B1) and/or B2), or a recombinant microorganism containing the expression cassette described in B3), or a recombinant microorganism containing the recombinant vector described in B4); B6) a transgenic plant cell line comprising the nucleic acid molecule described in B1) and/or B2), or a transgenic plant cell line comprising the expression cassette described in B3), or a transgenic plant cell line comprising the recombinant vector described in B4); B7) a transgenic plant tissue containing the nucleic acid molecule of B1) and/or B2), or a transgenic plant tissue containing the expression cassette of B3); B8) A transgenic plant organ containing the nucleic acid molecule of B1) and/or B2), or a transgenic plant organ containing the expression cassette of B3). 4. application according to claim 3, is characterized in that, described nucleic acid molecule is following any one: C1) the coding sequence is the DNA molecule shown in SEQ ID No.2 or positions 1-204 of SEQ ID No.2; C2) The nucleotide sequence is the DNA molecule shown in SEQ ID No. 2 or positions 1-204 of SEQ ID No. 2. 5. A method for cultivating a transgenic plant, characterized in that the method comprises increasing and/or reducing the content and/or activity of the protein described in claim 1 or 2 in the target plant to obtain the transgenic plant. 6. method according to claim 5 is characterized in that, described improving the content and/or activity of the protein described in claim 1 or 2 in the target plant by improving the expression level of the gene encoding the protein in the target plant accomplish. 7. method according to claim 6, is characterized in that, described improving the expression level of the coding gene of the protein described in the plant of interest is realized by introducing the coding gene of the protein described in claim 1 or 2 into the plant of interest . 8. method according to claim 5 is characterized in that, described reducing the content and/or activity of the protein described in claim 1 or 2 in the target plant by reducing the expression level of the gene encoding the protein in the target plant accomplish. 9. method according to claim 8, is characterized in that, described reducing the expression level of the coding gene of described protein in the target plant is to utilize gene mutation, gene knockout, gene editing or gene knockdown technology to make the target plant genome. The activity of the gene encoding the protein described in claim 1 or 2 is decreased or inactivated. 10. The protein of claim 1 or 2, and/or the biological material of claim 3.

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