CN104805100A - Application of paddy rice gene OsS[mu]BP-2 in delaying plant leaf senescence - Google Patents

Abstract

The invention discloses the application of the rice gene OsSμBP-2 in delaying plant leaf senescence. The nucleotide sequence of the rice gene OsSμBP-2 is shown in SEQ ID NO.1. The application includes: connecting the rice gene OsSμBP-2 into an expression vector to construct a recombinant expression vector; transforming the recombinant expression vector into a recipient plant. The present invention clones the gene OsSμBP-2 from the premature aging mutant ossμbp-2 of rice by Tula cloning technology; and proves that the gene OsSμBP-2 can delay the senescence of plant leaves through functional complementation experiments; the gene OsSμBP-2 can be applied to the field of plant breeding , which is of great significance to the screening and breeding of aging-resistant plant varieties.

Description

Application of rice gene OsSμBP-2 in delaying plant leaf senescence

technical field

The invention relates to the technical fields of plant genetic engineering and rice molecular breeding, in particular to the application of a rice gene OsSμBP-2 in delaying plant leaf senescence.

Background technique

Leaf senescence is a manifestation of plant adaptation to the environment and is a necessary stage of growth and development. The senescence process is usually accompanied by the reorganization of carbohydrates and the transfer of related substances to young leaves or seeds, which is of great significance to the reproduction of offspring (NamH G.The molecular genetic analysis of leaf senescence. Curr Opin Biotechnol, 1997, 8: 200-207). However, the premature senescence of functional leaves during the growth and development of rice, especially at the filling and heading stage, will lead to low seed setting rate, high empty seedling rate and poor quality. Studies have shown that the senescence of functional leaves at the filling and heading stage of rice can be increased by about 2% in theory, and about 1% in practice (Liu Daohong. Senescence of plant leaves. Plant Physiology Communications, 1993, 2: 14-19; Ma Yuefang and Lu Dingzhi. Effects of irrigation methods on senescence and some physiological activities in the late growth period of hybrid rice, Chinese Rice Science, 1990, 2: 56-6), and can also improve rice quality (ThomasH and Smart CM.Crops that stay green.Ann Appl Biol, 1993, 123:193-219). Hybrid rice has the characteristics of well-developed root system, strong tillering ability, large leaf area, large panicle and many grains, high yield and high quality, etc. It has been widely used in my country and has made an important contribution to ensuring food security in my country. However, some hybrid rice, especially hybrid indica rice (such as Liangyoupeijiu) is more prone to premature senescence in the later stage, which will lead to a rapid decline in the dry matter production rate in the later stage, which will affect the grain filling process and ultimately affect the yield potential. In recent years, although scientists have made great efforts to solve this problem through traditional and modern breeding methods and have achieved remarkable results, the problem of premature leaf senescence in hybrid indica rice is still an important problem that rice genetics and breeders need to solve urgently.

Regarding the causes of aging, a lot of researches have been done from the perspective of leaf morphology and molecular physiological characteristics in the past few decades, and put forward the theory of free radical damage, gene regulation, light-carbon imbalance, nutritional stress and hormone balance, etc. Theoretical hypothesis (Wei Daozhi, Dai Xinbin, Xu Xiaoming. Several hypotheses on the mechanism of plant leaf aging. Guangxi Plants, 1998, 18: 89-96; Brutovska E, Samelova A, Dusicka J and Micieta K. Aging of trees: Application of general aging theories. Aging Research Reviews, 2013, 12: 855-866). Although these hypotheses have made reasonable explanations for the senescence initiation and senescence process of leaves to a certain extent, there is still a certain distance from the real solution to the mechanism of senescence, and more in-depth and detailed research is needed. The reason is that each of the hypotheses one-sidedly emphasizes some factors that cause leaf senescence. Among them, the gene regulation theory emphasizes the importance of the sequence and stage expression of genes in the growth and development; the light-carbon imbalance theory focuses on the transmission of light energy, Utilization and active oxygen damage; nutritional stress theory focuses on the supply-demand balance between sources and sinks; hormone balance theory focuses on the relationship between underground and aboveground and the balance between different hormones. However, leaf senescence is not determined by simple factors. During the aging process, senescent cells undergo significant changes in structure, physiological and biochemical changes, and molecular levels. It is a joint regulation involving many factors such as ions, hormones and genes. complex system process.

It is now basically clear that abscisic acid, salicylic acid and jasmonic acid are important hormones that promote plant leaf senescence; while Ca ²⁺ , K ⁺ and N are considered to be mineral nutrients that are closely related to leaf senescence in crops such as rice and cotton element; used by Liu et al. The sword leaves of Liangyoupeijiu were used as materials, and 533 aging-related differentially expressed genes were identified by suppression subtractive hybridization. Skeleton formation and flower development, etc. Wu et al. (Wu X Y, Kuai B K, Jia J Z, Jing H C. Regulation of leaf senescence and crop genetic improvement. J Integr Plant Biol, 2012, 54: 936-952) divided leaf senescence-related genes into 7 categories according to gene function, Including transcription factors, protease or kinase genes, genes involved in metabolic processing and degradation, material transport related genes, genes involved in catalysis, binding protein genes and biosynthesis related genes.

SμBP-2 was first cloned in the human genome (Mizuta TR, Fukita Y, Miyoshi T. Shimizu A, Honjo T. Isolation of cDNA encoding a binding protein specific to 5'-phosphorylated single-stranded DNA with G-rich sequences. Necleic Acids Res.1993, 21: 1761-1766), studies suggest that it belongs to Upf1-like helicase (Jankowsky E. RNA helicases at work: binding and rearranging. Trends Biochem Sci. 2011, 36: 19-29), the gene Mutations will cause type I distal spinal muscular atrophy in humans (LimSC, Bowler MW, Lai TF, Song H. The Ighmbp2helicase structure reveals the molecular basis for disease-causing mutations in DSMAl. Nucleic Acids Res. 2012, 40: 11009-92 ). The function of this type of gene in rice has not been involved so far.

Rice is one of the most important food crops, and more than half of the world's population depends on rice as a staple food. In our country, with the continuous increase of population and decrease of land area, the demand for increasing rice production is becoming more and more urgent, and the national food security strategy is particularly important. Therefore, it is of great significance to cultivate aging-resistant rice. However, there is no drought-resistant and senescence-resistant transgenic rice in rice at present, so it is of great significance to find and clone senescence-resistant genes and breed senescence-resistant varieties to increase rice yield and improve rice quality.

Contents of the invention

The invention provides the application of a rice gene OsSμBP-2 in delaying the senescence of plant leaves, and the gene can delay the senescence of plant leaves.

Application of the rice gene OsSμBP-2 in delaying plant leaf senescence, the nucleotide sequence of the rice gene OsSμBP-2 is shown in SEQ ID NO.1.

The rice gene OsSμBP-2 belongs to Upf1-like helicase gene, so it is named OsSμBP-2. During rice leaf senescence, OsSμBP-2, as a negative regulator, can delay leaf senescence. Using the promoter of the leaf senescence-specific expression gene to promote the expression of the OsSμBP-2 gene and transform the plant can delay the occurrence of leaf senescence in the later stage of plant reproductive growth and improve the photosynthesis of the plant. The amino acid sequence of the protein encoded by the rice gene OsSμBP-2 is shown in SEQ ID NO.2.

Specifically, the applications include:

(1) connecting the rice gene OsSμBP-2 into an expression vector to construct a recombinant expression vector;

(2) Transforming the recombinant expression vector into recipient plants.

Wherein, the recipient plant may be rice or Arabidopsis, preferably, the recipient plant is rice.

The present invention also provides a recombinant expression vector, including an original vector and a target gene inserted into the original vector, the base sequence of the target gene is shown in SEQ ID NO.1.

The construction method of the recombinant expression vector is a conventional method. Preferably, the original vector in the recombinant expression vector is pCAMBIA1300 or pSB326-Actin-NOS. Among them, pSB326-Actin-NOS is an overexpression vector, which can significantly increase the expression of the target gene.

The invention also provides a transformant comprising the recombinant expression vector. When transforming the recipient plant, an Agrobacterium-mediated transformation method can be used, specifically, the Agrobacterium is Agrobacterium EHA105.

Compared with the prior art, the present invention has the following beneficial effects:

The present invention clones the gene OsSμBP-2 from the premature aging mutant ossμbp-2 of rice by Tula cloning technology; and proves that the gene OsSμBP-2 can delay the senescence of plant leaves through functional complementation experiments; the gene OsSμBP-2 can be applied to the field of plant breeding , which is of great significance to the screening and breeding of aging-resistant plant varieties.

Description of drawings

Fig. 1 is the phenotype of mutant ossμbp-2 of the present invention at different growth stages;

(A) tillering stage; (B) heading stage;

Fig. 2 is the result of OsSμBP-2 gene mapping and sequencing analysis of the present invention;

(A) Initial gene mapping; (B) Gene fine mapping; (C) BAC in the localization interval; (D) ORF in the localization interval; (E) Structure and mutation site of the mutant gene; (F) Wild-type locus Sequencing map; (G) The sequencing map of the mutation site of the mutant; the mutation site is shown by the arrow;

Fig. 3 is the verification result of genetic complementation of the mutant ossμbp-2 of the present invention.

Detailed ways

The molecular biology and biochemical methods used in the following examples are all known techniques, written by Ausubel in Current Protocols in Molecular Biology published by John Wiley and Sons company and written by J.Sambrook etc. by Cold Spring Harbor Laboratory Press (2001) published Molecular Cloning: A Laboratory Manual, 3 ^rd ED. and other documents have detailed instructions. The experimental materials used in the following examples are commercially available products unless otherwise specified.

Example 1 Isolation and genetic analysis of mutants

The indica rice variety Zhehui 7954 was mutated by EMS, and the leaf premature senescence mutant ossμbp-2 was screened out from the progeny, and a line with stable mutant traits was obtained through multiple generations of backcrossing and selfing. The premature senescence trait started at the tillering stage of the ossμbp-2 mutant plants (Fig. 1A), and at the heading and flowering stage, all leaves showed different degrees of premature senescence symptoms (Fig. 1B). Using ossμbp-2 as _the female parent, F ₁ was obtained by crossing with Zhehui 7954 and japonica rice material ₀₂₄₂₈ respectively. The F ₁ plants appeared normal; The segregation ratio wild type: mutant = 3:1, thus indicating that the senescence trait is controlled by a single recessive gene.

Example 2 Fine mapping of aging gene OsSμBP-2

Using the F ₂ population of ossμbp-2/02428 as the mapping population, a total of 215 premature aging individuals were obtained. 500 pairs of SSR primers and InDel markers evenly distributed on 12 rice chromosomes were selected to analyze the polymorphism of the japonica rice material 02428 and the mutant ossμbp-2 one by one, and the normal gene pool and the mutant gene pool were analyzed using the screened polymorphic primers . It was found that the SSR markers R3M23 and RM168 on rice chromosome 3 had a significant partial separation between the mutant gene pool and the normal gene pool. The above markers were verified by using 215 progeria individual plants, and OsSμBP-2 was preliminarily located on RM168 and RM168. between R3M23 (Fig. 2A).

Another 20 pairs of primers were searched and designed near R3M23 and RM168, 6 of which showed good polymorphism between the parents, which were SSR markers RM5488, RM3646, RM15361, RM6266, RM3513, RM15363 and InDel marker R3M30. With 7 pairs of markers, OsSμBP-2 was finally located between RM15363 and RM15361 (Fig. 2B), with a physical distance of 121kb, two BACs in the region, and a total of 14 annotated genes (Table 1, Fig. 2C and 2D).

Table 1 Molecular markers for fine mapping of OsSμBP-2 gene

      

Example 3 Prediction of aging mutation genes and comparative analysis of sequencing

According to the positioning results in Example 2, after functional analysis of the 12 annotated genes in this interval, 3 of the candidate genes were sequenced using 12 pairs of specific primers, and it was found that a base occurred in the ORF region of the candidate gene LOC_Os03g38990 (G) (Fig. 2F) is mutated to A (Fig. 2G), which in turn causes changes in the splicing of the mature mRNA of the gene, and a frameshift mutation in the amino acid coding region. In order to further verify the existence of the mutation site, we sequenced the normal phenotype and progeria phenotype individual plants in the F2 generation of the mapping population, and found that the progeria phenotype individual plants all had the mutation site, while the normal phenotype individual plants strains do not have this mutation site.

Example 4 Verification of genetic complementation of OsSμBP-2

Construction of gene complementation vector: Design a pair of primers according to the sequence of the full-length cDNA (GenBankaccession number: AK099721) of the OsSμBP-2 gene in GenBank:

Upstream primer: OsSμBP-2F: 5'-ATGGCGGGGCGGAGTGGC-3';

Downstream primer: OsSμBP-2R: 5'-CAGT GGTACC TCAGCTCTGGTATTCTGAT-3' (contains KpnI restriction site);

The cDNA of OsSμBP-2 gene was amplified from Nipponbare by RT-PCR technology, and the OsSμBP-2-cDNA fragment was obtained by electrophoresis purification;

Simultaneously use the following primers with the aid of the high-fidelity enzyme PrimerSTAR:

OsSμBP-2PF: 5'-CAGT TCTAG ATAGCCACAGAACCGTTAGTC-3' (contains the restriction site of XbaI);

OsSμBP-2PR: 5'-GCCACTCCGCCCCGCCAT-3';

PCR reaction system:

      

Reaction parameters: Denaturation at 98°C for 10 seconds, annealing at 55°C for 15 seconds, extension at 72°C for 2 minutes, a total of 35 cycles.

Extension at 72°C for 5 minutes.

Amplify the Nipponbare genome to obtain the promoter of the OsSμBP-2 gene, and purify it by electrophoresis to obtain the OsSμBP-2-Promoter fragment; then use fusion PCR technology to fuse the OsSμBP-2-cDNA fragment with the OsSμBP-2-Promoter fragment and use the primer OsSμBP -2PF and OsSμBP-2R were expanded to finally obtain the OsSμBP-2 fragment containing the promoter and cDNA, and the purified PCR product was digested with KpnI and XbaI, and connected into the Agrobacterium plasmid pCAMBIA1300-Nos to form pC1300-OsSμBP-2 , select the plasmid with the correct insert for sequencing to determine the correct insert. At the same time, Agrobacterium-mediated transformation of Agrobacterium EHA105 was used to transform the mutant ossμbp-2.

Using Agrobacterium-mediated genetic transformation, the gene and complementation vector pC1300-OsSμBP-2 were respectively introduced into the mutant ossμbp-2 to obtain transgenic plants and planted in the field. The results showed that the transgenic offspring of pC1300-OsSμBP-2 returned to normal without premature aging ( FIG. 3 ), thus confirming that the candidate gene was the target gene.

Example 5 Cultivation of anti-senescence transgenic rice without marker gene

Gene overexpression vector construction: Design a pair of primers according to the sequence of the full-length cDNA (GenBankaccession number: AK099721) of the OsSμBP-2 gene in GenBank:

Upstream primer OsSuBP-2-1F: 5′-CAG TCTAGACC ATGGCGGGGCGGAGTGGC-3′ (contains the restriction site of XbaI);

Downstream primer OsSμBP-2-1R: 5′-CAGT GGTACC TCAGCTCTGGTATTCTGAT-3′ (contains KpnI restriction site);

The OsSμBP-2 gene was amplified from the plasmid pC1300-OsSμBP-2 in Example 4 by PCR technology, and the OsSμBP-2 fragment was obtained by electrophoresis purification, and the OsSμBP-2 fragment was double digested with XbaI and KpnI, and inserted into the pSB326-Actin-OsSμBP-2-NOS was obtained from the KpnI-digested over-standard expression vector pSB326-Actin-NOS, and the correct inserted plasmid was selected for sequencing to determine the correct inserted fragment.

PCR reaction system:

      

Reaction parameters: Denaturation at 98°C for 10 seconds, annealing at 55°C for 15 seconds, extension at 72°C for 2 minutes, a total of 35 cycles.

Extension at 72°C for 5 minutes.

The method for obtaining transgenic rice is to adopt existing technology (PanG., et al., Map-based cloning of novel rice cytochrome P450genes Cyp81A6that confers resistance to two different classes of herbicides. Plant Molecular Biology, 2006, 61: 933-943). The plump seeds of rice variety Nipponbare were dehulled and induced to produce callus as transformation material. The T-DNA vector pSB326-Actin-OsSμBP-2-NOS obtained in Example 3 was introduced into Agrobacterium EHA105 by electric shock method. Take the Agrobacterium plate containing the T-DNA vector pSB326-Actin-OsSμBP-2-NOS, pick a single colony and culture it in LB medium to prepare Agrobacterium for rice transformation.

Blot the rice callus to be transformed dry on sterile filter paper, put the callus into the Agrobacterium bacterium solution with OD ₆₀₀ of 0.5 (containing acetosyringone, 200 μmol/L), and place it at room temperature for 40 minutes Discard the bacteria solution, then place the rice callus on sterile filter paper to absorb excess bacteria solution, transfer the callus to co-culture medium for 50-55 hours, and transfer the callus without many Agrobacteria on the surface into Cultivate on the bacteriostatic medium for 5-7 days, and then transfer the callus without Agrobacterium on the surface to culture on the selection medium for 6 weeks (subculture once every two weeks). Transfer the resistant callus obtained after screening to the pre-differentiation medium (first cultured in the dark for 5-7 days, then 16 hours of light differentiation and germination) for 4-6 weeks, and transfer to the rooting medium after the resistant seedlings grow up After rooting, the regenerated plants are washed and cultivated in the greenhouse or in the field until the seeds are harvested. Nos terminator and primers of hygromycin phosphotransferase gene (hereinafter referred to as HPH gene) were used to identify the T0 generation plants, and the seeds on the T0 generation plants containing OsSμBP-2 gene and HPH gene were harvested. The seeds on the plants of the T0 generation were planted into the transgenic seedlings of the T1 generation, the Nos terminator and the HPH gene primer were used to identify the transgenic seedlings, and the seeds of the transgenic seedlings with only the OsSμBP-2 gene were selected and harvested.

After transplanting the seedlings of the above-mentioned transgenic rice seeds and the rice seeds without the OsSμBP-2 gene into the field, observe and detect the growth of the leaves at the tillering stage and the heading and flowering stage, and it is found that the rice overexpressing the OsSμBP-2 gene is at the heading 28 The total amount of chlorophyll in Tianhou flag leaf was significantly higher than that of the control, reaching 14.13% (Table 2), which indicated that the gene had the function of delaying leaf senescence.

Table 2 Chlorophyll content of flag leaf on the day of heading and 28 days after heading of overexpression transgenic plants and controls

      

Claims (8)

1. paddy gene OsS μ BP-2 is delaying the application in plant leaf aging, it is characterized in that, the nucleotide sequence of described paddy gene OsS μ BP-2 is as shown in SEQ ID NO.1.

2. apply as claimed in claim 1, it is characterized in that, comprising:

(1) described paddy gene OsS μ BP-2 is connected in expression vector, builds and obtain recombinant expression vector;

(2) by described recombinant expression vector transformation receptor plant.

3. apply as claimed in claim 2, it is characterized in that, described recipient plant is paddy rice.

4. a recombinant expression vector, comprise the goal gene of initial carrier and the described initial carrier of insertion, it is characterized in that, the base sequence of described goal gene is as shown in SEQ ID NO.1.

5. recombinant expression vector as claimed in claim 4, it is characterized in that, described initial carrier is pCAMBIA1300 or pSB326-Actin-NOS.

6. one kind comprises the transformant of recombinant expression vector described in claim 4 or 5.

7. transformant as claimed in claim 6, it is characterized in that, Host Strains is Agrobacterium.

8. transformant as claimed in claim 7, it is characterized in that, described Agrobacterium is Agrobacterium EHA105.