CN119432867A

CN119432867A - CiSPL3 gene of Carya vesiculata and its application in regulating flowering time and stress tolerance of plants

Info

Publication number: CN119432867A
Application number: CN202411330382.4A
Authority: CN
Inventors: 王敏; 朱灿灿; 陈于
Original assignee: Institute of Botany of CAS; Shanghai Academy of Agricultural Sciences
Current assignee: Institute of Botany of CAS; Shanghai Academy of Agricultural Sciences
Priority date: 2024-05-31
Filing date: 2024-09-24
Publication date: 2025-02-14

Abstract

The invention discloses a apocarya CiSPL gene, wherein the nucleotide sequence of the CiSPL gene is shown as SEQ ID NO. 1. The CiSPL provided by the invention is an important gene affecting the flowering early and late of apocarya and drought and salt stress resistance, and the over-expression CiSPL gene enables the flowering time of plants to be early, the drought resistance to be enhanced, the salt stress sensitivity to be enhanced, and the identification and application of SPL gene families to be expanded.

Description

Apocarya CiSPL gene and application thereof in regulating flowering time and adversity stress capability of plants

Technical Field

The invention relates to an application of CiSPL gene separated from apocarya, in particular to an application of apocarya CiSPL gene in regulating and controlling plant flowering time and adversity stress, belonging to the technical field of plant genetic engineering

Background

Carya illinoensis [ Carya illinoinensis (Wangehn.) K.Koch ] is a plant of the genus Carya of the family Juglandaceae, also known as Carya illinoensis, pecan, and pecan, which has large nut size, thin shell, high yield, good color, taste, smell, no astringency, and rich nutrition, and is an ideal health food. Apocarya is also an important woody oil tree species, the kernel oil content exceeds 70%, the unsaturated fatty acid content is up to 97%, and the apocarya is shelf-stable and is a superior edible oil. Along with the economic development and the improvement of the living standard of people, the demand of the apocarya on the market at home and abroad is increased, and the apocarya industry has huge market prospect. At present, large-scale popularization and planting are performed in the adaptive areas of Jiangsu, yunnan, zhejiang, anhui and other provinces.

The apocarya has long nutritional growth period, late flowering and fruiting, low early yield and poor economic benefit, and is a bottleneck for restricting the industrial development of apocarya. The young apocarya (grafted seedling) needs 4-5 years to realize fruiting (the yield is only 2.8-3.5 kg/mu), and the single plant yield and economic benefit in the first 10 years are far lower than those of the walnut of the same age in 7-8 years and 10-12.5 kg/mu. Therefore, in order to improve early yield and benefit of apocarya planting and promote the rapid development of industrial health, it is highly required to regulate and control the balance relation between vegetative growth and reproductive growth, promote the transformation of vegetative growth into reproductive growth (flower formation transformation), start the expression of flower formation factors, shorten the fruiting period, promote the differentiation of flower buds and improve the fruiting quantity. Drought stress is one of the most prominent abiotic stresses in plants. Drought stress negatively affects the morphology, development, metabolism, and physiological regulation of plants in all aspects. The vast area of the south through which the China passes belongs to seasonal arid regions, and mainly comprises Jiangsu, anhui, zhejiang, jiangxi, hunan, hubei, sichuan and other provinces in the Yangtze river basin. The seasonal drought in these areas has high frequency and intensity, and the drought and heat are basically synchronous, the evaporation capacity is large, and the physiological metabolism, growth and development, fruit yield and quality of the economic trees are seriously affected. The apocarya is easily and strongly influenced by water deficiency in the growth and development process, so that the introduction area can ensure the normal growth and obtain better fruit yield and quality only by meeting the water requirement of the apocarya.

SPL transcription factors are a family of genes specific to plants and are widely found in plants. The SPL gene family is widely involved in regulating plant growth and development and responding to adversity stress. Overexpression of switchgrass SPL7 and SPL8 can promote flowering, and downregulation of SPL7 or SPL8 gene expression alone can delay flowering to some extent. ZmSPL13 and ZmSPL29 can significantly promote corn flowering, and have certain functional redundancy. SPL is a key regulator of stress response in plants. The miR156/SPL module regulates salt tolerance by upregulating MdWRKY in apples. OsSPL10 variation confers drought tolerance by directly regulating the expression of OsNAC2 and the production of ROS in rice. Most of the reported conservative SPL function studies at present are mainly concentrated in arabidopsis thaliana, rice equation plants. In the economic forest tree Carya illinoensis, SPL gene function research has not been reported yet.

Disclosure of Invention

The invention aims to provide CiSPL genes affecting the flowering time and the adversity stress capability of the apocarya, ciSPL is an important gene affecting the flowering early and late of the apocarya and the drought and salt stress tolerance capability, and the overexpression CiSPL genes enables the flowering time of plants to be early, the drought tolerance capability to be enhanced and the sensitivity to salt stress to be enhanced.

In order to solve the technical problems, the invention discloses a apocarya CiSPL gene, wherein the nucleotide sequence of the CiSPL gene is shown as SEQ ID NO. 1:

The invention also provides a protein coded by the CiSPL gene, and the amino acid sequence of the protein is shown as SEQ ID NO. 2:

Further, the invention also provides a primer pair for amplifying the CiSPL gene, which comprises CiSPL-F and CiSPL-R, wherein the nucleotide sequence of CiSPL-F is shown as SEQ ID NO.3, and the nucleotide sequence of CiSPL-R is shown as SEQ ID NO. 4.

Meanwhile, a recombinant expression vector, an expression kit, a transgenic strain or recombinant bacteria containing the CiSPL gene are also within the protection scope of the invention.

The application also provides a promoter of the apocarya CiSPL gene, the sequence of which is shown in SEQ ID NO. 9:

CCTACTCTGAATCTATCATTACTATTTTTGAACATACTCGTAAGTAGAAACCCATGA

AATACATTGAGCAAATGTGGTAGAGAATTAAGTCACAGTAGCGCAGCCGGCCTAT

AAAGTAAAAATTATTCACGTTACTATTCAGACCCATTGGTATTGTGCTTTTGAAGC

CAATGGTGATGCTTGCTTTCTACCCGGACCTCACAATTATTCTTACGCTGACAGGG

GGAAAATGGAGTTGGTGGTCCTCGCATAAGTTCTTCTCCTTTTCCAACTTTCTCGA

GGTTATTTTTGCCTTCAAAATCCTCTTCACTGCTGCTTGCTAACTCCATCTGTCCCCTTGTCTGTCTACTCTTGTTTTGCCAAA(SEQ ID NO.9)。

The application also provides a cloning method of the promoter, and the sequence used for cloning the promoter is as follows:

an upstream primer TGCCTGCAGGTCGACTCTAGATAAGCATATTGGTACGTGTGCTC;

and a downstream primer GGACTGACCACCCGGGGATCCTTTGGCAAAACAAGAGTAGACAG.

In a specific embodiment, the CiSPL gene is connected with a PHB vector to obtain a PHB-CiSPL3 overexpression vector, then the PHB-CiSPL expression vector is transformed into escherichia coli DH5 alpha competent cells, amplification and screening are carried out to obtain the PHB-CiSPL3 overexpression vector with correct sequencing, the PHB-CiSPL overexpression vector is further transformed into agrobacterium GV3101, and finally the GV3101-PHB-CiSPL engineering bacteria are obtained.

The invention further provides the apocarya CiSPL gene, protein coded by CiSPL gene, or recombinant expression vector containing CiSPL gene, an expression kit, recombinant bacteria and transgenic strain, and application of the promoter in regulating and controlling plant flowering time and adversity stress.

Specifically, the flowering of the regulation plant is that the apocarya CiSPL gene is over-expressed in the plant, and the obtained transgenic plant has the advantages of early flowering time, enhanced resistance to drought stress and reduced tolerance to salt stress.

Preferably, the plant is apocarya or Arabidopsis thaliana.

In the application process, the method comprises the following steps:

1) Providing the pecan of claim 1 CiSPL gene;

2) Connecting the apocarya CiSPL gene with a vector to obtain a recombinant vector;

3) Transferring the recombinant vector into agrobacterium to obtain recombinant agrobacterium;

4) And infecting the plant with the recombinant agrobacterium to obtain a plant over-expressing the apocarya CiSPL gene.

The CiSPL gene provided by the invention is an important gene affecting the flowering early and late of apocarya and drought and salt stress resistance, and the over-expression CiSPL gene enables the flowering time of plants to be early, the drought resistance to be enhanced, the salt stress sensitivity to be enhanced, and the identification and application of SPL gene families to be expanded.

Drawings

FIG. 1 plant SPL protein phylogenetic analysis;

FIG. 2 shows the expression of CiSPL genes in different tissues of apocarya and at different stages of bud differentiation of apocarya;

FIG. 3 is a functional analysis of CiSPL genes in Arabidopsis, A: ciSPL gene overexpression in Arabidopsis can significantly advance plant flowering, B: ciSPL3 transgenic Arabidopsis and wild plant flowering days analysis;

FIG. 4 is a diagram showing the result of electrophoresis of CiSPL gene promoter PCR amplification (primer with restriction enzyme sites);

FIG. 5 is a CiSPL gene promoter cis-acting element analysis;

FIG. 6 shows the activity analysis of the promoter of the GUS staining test CiSPL gene in different tissues of Arabidopsis;

FIG. 7 shows the functional analysis of CiSPL genes in Arabidopsis, A. Growth of wild type and transgenic Arabidopsis seedlings in MS medium containing 300mM mannitol, B. Root length statistics after seedling germination, C. Plant phenotype after drought treatment;

FIG. 8 shows functional analysis of CiSPL genes in Arabidopsis, A. Phenotype results of wild-type and transgenic Arabidopsis seedlings 15 days after germination in MS medium containing 100mM and 200mM NaCl, B. Root length statistics after seedling germination, C. Phenotype of wild-type and transgenic Arabidopsis lines 20 days on MS medium containing 100mM NaCl, D. Phenotype of plants under 300mM NaCl treatment.

Detailed Description

The foregoing and/or other advantages of the invention will become more apparent from the following detailed description of the invention when taken in conjunction with the accompanying drawings and detailed description.

Cloning of the CiSPL genes of example 1.

According to the cds sequence of the gene CiSPL in the genome database of apocarya, the cDNA of the female flower bud of apocarya is used as a template, and the CiSPL gene is obtained by cloning.

Specifically, female flower buds of 7-8-year-old apocarya are collected, transZol Up Plus RNAKit kit is used for extracting total RNA of female flower buds of apocarya, and the female flower bud RNA is reversely transcribed into cDNA according to the step of PRIMESCRIPTTM RT REAGENT KIT (Takara) reverse transcription kit. Designing amplification primers based on CDS sequence of apocarya gene published by apocarya genome (Carya illinoinensis Pawnee v 1.1.1) at Phytozome 13.0.0,

The upstream primer CiSPL-F (SEQ ID NO. 3): ATGGAAACAGGCAGAGCTGAGGG;

the downstream primer CiSPL-R (SEQ ID NO. 4): TCAATGTGAACCTTCTCCATAAG.

PCR amplified products were detected by 1% agarose gel electrophoresis and photographed in a gel imaging system. And cutting off a DNA target band, recovering and purifying the target fragment by using a gel recovery kit, connecting the target fragment to a pMD19-T vector to obtain a pMD19-CiSPL recombinant vector, then transforming E.coli DH5 alpha competent cells, and taking positive clones and then sending the positive clones to a company for sequencing. Wherein, the ligation reaction of the target gene is carried out on a PCR instrument, and the reaction system is pMD19-TSimple Vector 0.5.5. Mu.l, DNA fragment 4.5. Mu.l, solutionI. Mu.l. After mixing well, the mixture was placed in a PCR apparatus at a temperature of 16℃for about 10 hours or overnight. The transformation procedure was to add a total of 10. Mu.l of the system to 100. Mu.l DH5a E.coli competent cells and leave them on ice for 30 minutes. 42 ℃,2 minutes, and then 2-3 minutes on ice (during which the centrifuge tube is not shaken). 800. Mu.l of LB liquid medium (without antibiotics) was taken on an ultra-clean bench, added to the competent cell liquid, and shake cultured at 37℃for about 1 hour. Centrifugation is carried out at 37 ℃ for 5 minutes at 8000g, and the bacterial cells are precipitated. The most supernatant was discarded on a sterile bench, and about 100. Mu.l was left, and the cells were suspended by upward and downward blowing with a pipette and uniformly spread on LB solid medium containing Kana (Kan). After sealing with sealing film, the plate was placed in a 37℃incubator overnight for culturing until single colonies were grown. Single colony is picked into LB liquid culture medium added with Kana (Kan), shaking is carried out at 37 ℃ and 200rpm for about 5 hours, bacterial liquid is used as a template, bacterial liquid PCR detection is carried out, and if target fragments exist, bacterial liquid is sent to sequencing.

Cloning to obtain CiSPL gene sequence full-length 444bp, coding 147 amino acids. The base sequence of the nucleotide sequence is shown as SEQ

The amino acid sequence of the expressed protein is shown as SEQ ID NO.1 and SEQ ID NO. 2.

The relative molecular weight, theoretical isoelectric point, hydrophilicity, etc. of CiSPL3 were analyzed using an online website ExpASY (https:// web. ExPASy. Org/protparam /). The protein conserved domain was analyzed through NCBI Conserved Domain Search (https:// www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb. Cgi) website. Homologous sequences of CiSPL proteins were searched for in NCBI database BLAST, and phylogenetic tree was constructed using MEGA5.0 software.

On-line prediction CiSPL of protein molecular mass is 16.67kD, theoretical isoelectric point (pI) is 7.05, instability coefficient is 73.36, fatty acid amino acid index is 35.24, average hydrophilicity average value (GRAVY) is-1.227, so that it is presumed to be unstable hydrophilic protein. NCBI Conserved Domain Databas (CDD) analysis revealed that the CiSPL protein sequence contained an SBP domain between amino acids 63 to 137. Phylogenetic tree results of SPL3 proteins from different species show (FIG. 1) CiSPL. The same small branch as that of the walnut JrSPL protein is in a relatively close genetic distance with the walnut.

We studied the external morphology and anatomical structure of the female flower bud of Carya illinoensis in earlier stage, and divided them into 5 periods of FB1 (period of onset of female flower development from female flower development), FB2 (period of female flower sequence formation), FB3 (period of female flower primordium formation), FL1 (initial stage of flowering), FL2 (full-bloom stage). Then, transcriptome sequencing analysis is carried out on the female flower buds of the apocarya at different differentiation periods, as shown in fig. 2A, ciSPL genes are mainly subjected to continuous high expression in the process of transforming stem apical meristem into flower meristem (in an undifferentiated period, in an initial differentiation period and in an inflorescence formation period), namely, the expression is obviously up-regulated in the process of developing the flower buds into female flowers. CiSPL3 gene was expressed in each tissue of apocarya, where the expression level was significantly higher in female flowers and fruits than in other tissues, with the highest expression level in female flowers (FIG. 2B). The CiSPL gene provided by the application is expected to be applied to apocarya and is used for regulating and controlling flowering time and adversity stress capability of plants.

Example 2CiSPL construction of Gene overexpression vector.

Designing a primer containing an enzyme cutting site by taking a pMD19-CiSPL recombinant vector as a template:

BamHI(SEQ ID NO.5):

CTCTCTCTCAAGCTTGGATCCATGGAAACAGGCAGAGCTGAGGG;

PstI(SEQ ID NO.6):

GATCAATTCGAGCTCCTGCAGTCAATGTGAACCTTCTCCATAAG. PCR amplification was performed using KOD DNA polymerase (KOD-401, TOYOBO), 20. Mu.l of PCR reaction system 2. Mu.l of 10 Xbuffer, 2. Mu.l of 2mM dNTPs, 2. Mu.l of DNA template, 1. Mu.l of DMSO, 0.8. Mu.l of 10 pmol/. Mu.l of upstream and downstream primers, and 20. Mu.l of water was supplemented with 0.5U KOD DNA polymerase (KOD-401, TOYOBO). The PCR amplification procedure comprises 4min of pre-denaturation at 94 ℃,30 s of annealing at 50-64 ℃, 1min/kb of extension at 68 ℃,40 cycles and 8min of heat preservation at 68 ℃. And (5) DNA electrophoresis and recovery of target fragments.

The CiSPL fragment and PHB vector (purchased from Shanghai Pubrier Biotechnology Co., ltd.) were double digested with BamHI and PstI restriction enzymes, and the digested fragments were recovered and ligated into EasyGenoDNA recombinant system (#VI 201-02, tiangen organism) in 10ul of recombinant system as follows.5 ul of 2X EasyGeno Assembly Mix,2.5ul of digested vector DNA,2.5ul of fragment DNA. The reaction system is added into a 250ul EP tube, the escherichia coli coated plate is transformed after being put in a water bath at 50 ℃ for 30 minutes, the bacteria are picked up after being put in an incubator at 37 ℃ for 16 hours, sequencing is carried out, positive bacterial liquid is selected for sequencing confirmation, and finally the PHB-CiSPL3 super-expression vector is obtained. And transforming the PHB-CiSPL3 super-expression vector into agrobacterium GV3101 to finally obtain GV3101-PHB-CiSPL engineering bacteria. Specifically, the agrobacterium preserved at-80 ℃ is taken to be competent at room temperature and the live palm is immediately inserted into ice after part of the agrobacterium is melted and is in an ice water mixed state. Every 100 mu l of competent plasmid DNA is added with 0.1 mu g (volume is not more than 10 mu l), the mixture is stirred by hand at the bottom of the tube and then mixed well, and the mixture is placed on ice for 5 minutes, liquid nitrogen for 5 minutes, water bath at 37 ℃ for 5 minutes and ice bath for 5 minutes in sequence. 700 μl of LB liquid medium without antibiotics is added, and the culture is carried out for 2-3 hours at 28 ℃ with shaking at 200 rpm. After centrifugation at 6000rpm for one minute, about 100. Mu.l of supernatant was left to gently blow the resuspended pellet and spread on LB plates containing 50. Mu.g/ml kanamycin, and placed upside down in a 28℃incubator for 2-3 days. 1 single colony is randomly selected and used for colony PCR, and the correct agrobacterium single clone is identified and used for marking.

Example 3 functional identification of transgenic Arabidopsis thaliana.

The GV3101-PHB-CiSPL engineering bacteria obtained in example 3 are used for infecting wild Arabidopsis thaliana by adopting an inflorescence infection method, and T0 generation transgenic seeds 35S: ciSPL3 are obtained.

The inflorescence infection step comprises the steps of sterilizing wild arabidopsis seeds with a 5% sodium hypochlorite solution for 8 minutes, flushing the wild arabidopsis seeds with sterile water for 5 times, spreading the sterilized seeds in a plate containing an MS culture medium, vernalizing the seeds in a refrigerator at a temperature of 4 ℃ for 2-3 days, and placing the seeds in a climatic chamber to start germination and growth. And (3) selecting strong and consistent seedlings after germination for 7-12 days, transplanting the seedlings into culture soil soaked by the flower-lacking compound fertilizer in advance, covering the culture soil with a preservative film, and removing the seedlings after the seedlings survive.

The plants which are being bolting and flowering are watered fully one day in advance, the small pot is inverted, all inflorescences are inverted into the bacterial liquid which is suspended in advance for about 30 seconds, and the transformation is repeated once after 7 days according to the method. After 2-3 weeks, the nutrient solution is poured as little as possible to accelerate aging, mature seeds are collected in paper bags and are dried for 7 days.

And screening out resistant plants (30 mug/ml HYG) by using hygromycin resistance of the obtained T0 generation transgenic seeds, and simultaneously identifying by using a PCR method to obtain positive plants. After the positive plants obtain T1 seeds, the same method is respectively used until transgenic plants with stable inheritance of T3 generation are obtained. Three CiSPL transgenic lines, namely lines OE1, OE3 and OE10, are taken, wild arabidopsis thaliana and 35S: ciSPL3 transgenic T3 generation seeds are directly planted in a nutrition matrix (peat soil and vermiculite are uniformly mixed according to a ratio of 2:1), are cultured under normal conditions (16 h illumination/8 h darkness, a temperature of 22 ℃ and an illumination intensity of 150 mu mol/m ² s), and bolting and flowering time of each plant is observed.

As a result, as shown in FIG. 3, the flowering time of the three transgenic Arabidopsis lines (OE 1, OE3 and OE 10) was significantly advanced, about 5 days, compared to the wild type Arabidopsis, indicating that over-expression CiSPL has a function of promoting flowering of plants.

Example 4GUS staining assay CiSPL promoter activity of gene in different tissues of Arabidopsis thaliana.

Cloning of the promoter of the CiSPL gene:

the DNA of the apocarya 'Boni' is taken as a template, and the following primer sequences are adopted:

the upstream primer (with XbaI cleavage site, SEQ ID NO. 7):

TGCCTGCAGGTCGACTCTAGATAAGCATATTGGTACGTGTGCTC;

downstream primer (with BamHI cleavage site, SEQ ID NO. 8):

GGACTGACCACCCGGGGATCCTTTGGCAAAACAAGAGTAGACAG;

A sequence of 1500-2000bp upstream of the initiation codon (ATG) of CiSPL gene was amplified as a promoter, ciSPL was amplified to a 1994bp sequence as shown in FIG. 4, and the promoter sequence was shown as SEQ ID NO. 9. The resulting sequences were sequenced and aligned with the apocarya genomic data and were found to be all the expected promoter sequences.

The promoter sequence of the CiSPL gene was analyzed by online program PLANTCARE, and as shown in FIG. 5, the promoter sequence of CiSPL3 contains 18 functionally annotated cis-acting elements, namely ABRE, LTR, ARE, CGTCA-motif, RY-element, TGA-element, TGACG-motif, CAT-Box, TCT-motif, MBS, TC-RICH REPEATS, etc., 5 cis-acting elements related to light response, namely Box 4, G-Box, I-Box, G-Box, TCT-motif, which are widely present in promoters of plant genes. 9 cis-acting elements with other functions, 5 abscisic acid response related elements (ABRE), 4 MeJA hormone response elements (CGTCA-motif, TGACG-motif), 1 auxin response element (TGA-element), 1 low temperature response element (LTR), 2 drought induction elements (MBS), 1 defense and stress response cis-acting element (TC-RICH REPEATS), 1 zein metabolism regulatory element (O2-site), 3 anaerobic induction regulatory elements (ARE), 1 seed specific regulatory element (RY-element), 1 meristem expression regulatory related element (CAT-box).

Selecting a P1300GN vector, constructing a GUS fusion expression vector (CiSPL Pro:: GUS recombinant expression vector) of a apocarya CiSPL gene promoter, and transforming the constructed CiSPL Pro::: GUS recombinant expression vector into arabidopsis by using an agrobacterium-mediated inflorescence dip-dyeing method. Seeds of the T0 generation were sown on the resistance medium, and after two weeks, T1 generation transgenic seedlings with hygromycin resistance were obtained. The wild type Arabidopsis thaliana and CiSPL Pro are sown in a nutrition pot, the plant grows for about 35 days, leaves, roots, stem segments, flowers and fruit pods are taken for histochemical staining, and the result shows that the roots are deep in color, the expression is strong, the leaves are also dyed, but the leaves are lighter than the roots, the stems are light in color, the flower buds and the petals are deep in color, and the fruit pods are also dyed (figure 6).

Example 5 phenotypic observations and root length measurements in Arabidopsis seedlings under drought stress.

The sterilized wild type and transgenic Arabidopsis T3 generation seeds are respectively sown on an MS plate and an MS+300mM mannitol plate, vernalized for 2d under the dark condition at 4 ℃, and then vertically placed in a growth chamber for culturing at 23 ℃ under the illumination of 16h and the darkness of 8 h. And root length was measured after 15 days. As shown in FIG. 7 (A and B), the CiSPL gene OE3 strain was still significantly longer in seedlings than in wild-type Arabidopsis in mannitol-containing medium.

Wild type and transgenic potted seedlings of Arabidopsis grown normally at water for 5 weeks (35S: ciSPL3 transgenic plants) were drought treated for 15 days and observed for morphological characteristics. As a result, as shown in fig. 7C, under drought conditions, the wild-type control showed significant wilting 15 days after stress, and the transgenic lines showed wilting with water loss, but the symptoms were much lighter than the control, and bolting and flowering could be continued, and most of the transgenic lines began to revive after 3 days after the reversion to watering, while most of the wild-type lines still died. Thus, overexpression of CiSPL gene increases drought stress tolerance in Arabidopsis plants.

Example 6 phenotypic observations and root length measurements under salt stress in Arabidopsis thaliana.

The wild type and transgenic Arabidopsis thaliana T3 generation seeds were sterilized and then placed on MS, MS+100mM NaCl and MS+200mM NaCl medium, respectively. Meanwhile, the culture dish is vertically placed, after vernalization at 4 ℃ for 2 days, the culture dish is normally cultured in an illumination incubator, and after 15 days, the root length is counted and photographed. As shown in FIGS. 8A and 8B, in the salt stress medium, the root length of transgenic CiSPL gene Arabidopsis seedlings was significantly shorter than that of the wild type control.

After 20 days of growth of transgenic lines and Wild Type (WT) arabidopsis thaliana on NaCl (100 mM) containing medium, 76.2%, 78.6% and 52.4% of the plants, respectively, were completely albino dead, whereas the wild type had a survival rate of more than 80% (fig. 8C).

The wild type and transgenic lines of Arabidopsis grown normally at waterings were watered in 300mM NaCl solution for 5 weeks, once every three days, and repeated four times. After 15 days of salt stress treatment, the transgenic lines and wild-type plants exhibited significantly different trait manifestations. Leaves of the transgenic lines gradually died, and wild type plants also appeared to be different levels of dieback, but better than the transgenic lines (fig. 8D). In summary, overexpression of CiSPL gene significantly reduced salt stress tolerance in transgenic plants.

The invention provides CiSPL genes and ideas and methods for application thereof, and the method and the way for realizing the technical scheme are numerous, the above is only a preferred embodiment of the invention, and it should be pointed out that a plurality of improvements and modifications can be made to those skilled in the art without departing from the principle of the invention, and the improvements and modifications should be regarded as the protection scope of the invention. The components not explicitly described in this embodiment can be implemented by using the prior art.

Claims

1. A thin-shelled pecan gene, characterized in that the nucleotide sequence of the CiSPL3 gene is shown in SEQ ID NO.1.

2. The protein encoded by the CiSPL3 gene according to claim 1, whose amino acid sequence is shown in SEQ ID NO.2.

3. A primer pair for amplifying the CiSPL3 gene according to claim 1, characterized in that: CiSPL3 -F and CiSPL3 -R; the nucleotide sequence of the CiSPL3 -F is shown in SEQ ID NO.3; the nucleotide sequence of the CiSPL3 -R is shown in SEQ ID NO.4.

4. A recombinant expression vector, expression kit, transgenic strain or recombinant bacterium containing the CiSPL3 gene according to claim 1.

5. A promoter of the CiSPL3 gene of Carya syringae, characterized in that its sequence is shown in SEQ ID NO.8.

6. The promoter cloning method according to claim 5, characterized in that the sequence used for promoter cloning is:

Upstream primer: TGCCTGCAGGTCGACTCTAGATAAGCATATTGGTACGTGTGCTC;

Downstream primer: GGACTGACCACCCGGGGATCCTTTGGCAAAACAAGAGTAGACAG.

7. Use of the thin-shelled walnut CiSPL3 gene according to claim 1 or the protein encoded by the CiSPL3 gene according to claim 2, or the recombinant expression vector, expression kit, transgenic strain or recombinant bacteria according to claim 4, or the promoter according to claim 5 in regulating plant flowering time and adverse stress.

8. The use according to claim 5, characterized in that the regulating plant flowering is to overexpress the vegan walnut CiSPL3 gene in the plant, and the resulting transgenic plants have an early flowering time, enhanced resistance to drought stress, and reduced tolerance to salt stress.

9. The use according to claim 5, characterized in that the plant is Carya serrata or Arabidopsis thaliana.

10. The application according to claim 5, characterized in that it comprises the following steps:

(1) Providing the walnut CiSPL3 gene of claim 1;

(2) connecting the walnut CiSPL3 gene to a vector to obtain a recombinant vector;

(3) transferring the recombinant vector into Agrobacterium to obtain recombinant Agrobacterium;

(4) Infecting plants with the recombinant Agrobacterium to obtain plants that overexpress the CiSPL3 gene of Carya syringae.