Application of plant pigment interaction factor gene PIF4 in regulation and control of plant self-incompatibility

Abstract

The invention discloses application of a plant pigment interaction factor gene PIF4 in regulating and controlling plant self-incompatibility. The plant self-incompatibility can be effectively inhibited by inhibiting PIF4 gene expression. According to the invention, the fact that the plant pigment interaction factor gene PIF4 can regulate and control S-RNase expression is determined for the first time, so that the self-incompatibility of plants is regulated and controlled; the application is easy to operate and good in effect, the self-pollination maturing rate can be increased by 33%, and the method has excellent breeding application prospects.

Description

Technical Field

[0001] The present invention relates to the field of agronomy, and particularly to the application of the phytochrome interacting factor gene PIF4 in regulating plant self-incompatibility. Background Art

[0002] The reproductive isolation phenomenon caused by self-incompatibility in pears (Pyrus spp.) is the core biological bottleneck restricting their genetic improvement and efficient breeding. Self-incompatibility specifically recognizes and degrades homologous S-genotype pollen tubes through the stylar S-RNase protein, resulting in double fertilization failure. Although this mechanism ensures species genetic diversity, it seriously hinders the creation of pear self-inbred lines and the stable inheritance of excellent traits. In traditional cross-breeding, due to the high heterozygosity of self-incompatible genotypes, parental selection must strictly follow the S-genotype complementary principle, which not only greatly limits the selection range of cross combinations but also complicates the segregation pattern of target traits in the offspring population. Existing technologies rely on artificial pollination or physical and chemical treatments such as high temperature and high CO 2 to forcibly break through self-incompatibility barriers, but there are problems such as pollen viability loss, abnormal embryo development, and low phenotypic screening efficiency, making it difficult to meet the requirements of modern molecular breeding for high-purity parental lines and precise genetic manipulation.

[0003] The bud pollination technique achieves self-cross breakthrough by utilizing the window period of the dynamic change in the expression level of stylar S-RNase, revealing the key role of S-RNase spatio-temporal expression regulation in the conversion of self-compatibility. However, this technique has significant limitations in actual breeding applications: the pollination operation accuracy requirement in the small bud stage is extremely high, and the S-RNase expression level is affected by environmental factors (such as photoperiod and temperature fluctuations), showing significant phenotypic plasticity, resulting in poor genetic stability of the self-cross offspring population and making it difficult to establish a stable self-inbred line population.

[0004] In recent years, the research on plant transcription factor regulatory networks has provided new ideas for solving this problem. As an important member of the bHLH transcription factor family, PIF4 (Phytochrome Interacting Factor 4) has been found to have multiple functions of integrating light signals, temperature responses, and hormone regulation. Research has shown that PIF4 not only participates in plant photomorphogenesis, circadian clock synchronization, and flowering time regulation but also regulates the expression of downstream target genes by dynamically responding to environmental signals (such as red light inhibition and high temperature induction). However, currently, key scientific issues such as whether PIF4 participates in the regulation of pear S-RNase expression and how its molecular mechanism interacts with external environmental signals remain blank. Summary of the Invention

[0005] Object of the Invention: The object of the present invention is to provide the application of the phytochrome interacting factor gene PIF4 in regulating plant self-incompatibility.

[0006] Technical solution: The application of the phytochrome interacting factor gene PIF4 of the present invention in regulating plant self-incompatibility.

[0007] Preferably, the phytochrome interacting factor gene PIF4 is a gene having the sequence shown in SEQ ID NO: 1, or its homologous gene.

[0008] Preferably, the plant is a plant of the genus Pyrus in the Rosaceae family.

[0009] Preferably, the application is to inhibit the expression of the PIF4 gene to inhibit plant self-incompatibility.

[0010] Preferably, the steps of the application include:

[0011] (1) Design an inhibitory expression sequence according to the nucleotide sequence of the PIF4 gene, and construct a recombinant inhibitory vector;

[0012] (2) Introduce the recombinant inhibitory vector obtained in step 1 into the target plant.

[0013] Preferably, the inhibitory expression sequence in step 1 includes any one of a CDS characteristic sequence, miRNA, siRNA, sgRNA or an antisense strand.

[0014] Preferably, the inhibitory expression sequence is a CDS characteristic sequence, and the nucleotide sequence is as shown in SEQ ID NO: 2.

[0015] Preferably, the original vector of the recombinant inhibitory vector in step 1 is any one of pTRV2, pFGC5941, pYLCRISPR / dCas9-KRAB, pRS300, pBI121.

[0016] Preferably, the original vector of the recombinant inhibitory vector is pTRV2.

[0017] Preferably, step 2 is to introduce the recombinant inhibitory vector obtained in step 1 into the flower buds of the target plant.

[0018] Beneficial effects: Compared with the prior art, the present invention has the following remarkable advantages: 1. For the first time, it is clarified that the phytochrome interacting factor gene PIF4 can regulate the expression of S-RNase, and an application of the PIF4 gene in regulating self-incompatibility is provided; 2. This application is simple to operate and has good effects. The self-pollination seed setting rate can be increased by 33%, and it has excellent breeding application prospects. Description of the drawings

[0019] Figure 1 It is a RT-PCR detection result diagram of PIF4 in the style after inhibiting the phytochrome interacting factor gene PIF4;

[0020] Figure 2 RT-PCR detection result graph of S3-RNase in the style after inhibiting the phytochrome interacting factor gene PIF4;

[0021] Figure 3 Statistical result graph of the increased self-pollination seed setting rate after inhibiting the phytochrome interacting factor gene PIF4. Detailed implementation manners

[0022] The technical solution of the present invention will be further described below.

[0023] Example 1: Inhibiting the phytochrome interacting factor gene PIF4 can inhibit S-RNase expression

[0024] Using 'Cuiguan' pear as the target plant for experiments

[0025] (1) Based on the sequence of the phytochrome interacting factor PIF4 shown in SEQ ID NO: 1, designing the CDS characteristic sequence shown in SEQ ID NO: 2 and constructing it into the pTRV2 vector;

[0026] (2) Transforming the recombinant pTRV2 vector and the pTRV1 vector into Agrobacterium tumefaciens GV3101 respectively, screening to obtain Agrobacterium tumefaciens GV3101 containing the recombinant pTRV2 vector or the pTRV1 vector, and culturing until OD 600 = 1.0;

[0027] (3) After centrifugation and recovery, resuspending with infiltration buffer to a final concentration of OD 600 = 0.8,

[0028] wherein, the infiltration buffer contains 10 mM lithium chloride, 10 mM morpholineethanesulfonic acid and 200 μM acetosyringone;

[0029] (4) Mixing the Agrobacterium tumefaciens containing the pTRV1 vector and the Agrobacterium tumefaciens containing the recombinant pTRV2 vector or the non-recombinant pTRV2 vector by volume ratio of 1:1, and injecting into the flower buds at the bud sprouting stage, that is, about 3 days before flowering;

[0030] (5) After 3 days of treatment, collecting the styles, fully grinding under liquid nitrogen conditions, using TRIzol reagent to extract total RNA, and performing reverse transcription with HiScript II One Step RT-PCR Kit according to the instructions to obtain cDNA with a concentration of 0.02 μg / μL;

[0031] (2) Designing primers:

[0032] PIF4 forward primer: 5’-CTTCGTCTTCTGGCGGTTCT-3’;

[0033] PIF4 reverse primer: 5’-GATCCTGATCGATGCGCTGA-3’;

[0034] S3-RNase forward primer: 5’-ACATGGCTCCTGTGGGTATC-3’;

[0035] S3-RNase reverse primer: 5’-GTCCGGTTCAATCTTCGCCT-3’;

[0036] (3) The RT-PCR reaction system is shown in Table 1 and the reaction program is shown in Table 2, and RT-PCR detection is carried out.

[0037] Table 1 Reaction system

[0038] reactant system SsoFast EvaGreen supermix 5 μL Forward primer (10 μM) 0.5 μL Reverse primer (10 μM) 0.5 μL cDNA template 0.5 μL <![CDATA[ddH 2 O]]> 3.5 μL

[0039] Table 2 Reaction program

[0040]

[0041] The results are as Figure 1 、 2 shown. Compared with the non-recombinant pTRV2 vector treatment group (TRV), the recombinant pTRV2 vector treatment (TRV-PIF4) can effectively inhibit the expression of PIF4 and achieve the inhibition of S3-RNase.

[0042] Example 2: Inhibiting the phytochrome interacting factor gene PIF4 can inhibit the self-incompatibility of pears

[0043] (1) At the bud swelling stage, that is, 3 days before flowering, inhibit the expression of PIF4 by the method described in Example 1.

[0044] (2) Immediately after the flowers bloom, perform self-pollination. 100 flowers are pollinated in the non-recombinant pTRV2 vector treatment group and the recombinant pTRV2 vector treatment group respectively. Immediately after pollination, bagging is carried out, and 3 biological replicates are performed for each group.

[0045] (3) Check the fruiting situation after 15 days.

[0046] The results are as Figure 3 shown. The fruiting rate of the recombinant pTRV2 vector treatment group (41±6%) is significantly higher than that of the non-recombinant pTRV2 vector treatment group (8±3%), that is, inhibiting the phytochrome interacting factor gene PIF4 can improve the self-pollination fruiting rate while inhibiting the expression of S-RNase.

Claims

1. Application of the plant pigment interaction factor gene PIF4 in regulating plant self-incompatibility.

2. The use according to claim 1, characterized in that: The plant pigment interaction factor gene PIF4 is a gene having a sequence as shown in SEQ ID NO: 1, or a homologous gene thereof.

3. The use according to claim 1, characterized in that: The plant is a plant of the genus Pyrus in the Rosaceae family.

4. The use according to claim 1, characterized in that: The application is to inhibit the expression of PIF4 gene to inhibit the self-incompatibility of plants.

5. The use according to claim 4, characterized in that: The steps of the application include: (1) designing an expression inhibition sequence based on the nucleotide sequence of the PIF4 gene and constructing a recombinant inhibition vector; (2) Introducing the recombination suppression vector obtained in step 1 into the target plant.

6. The use according to claim 5, characterized in that: The expression inhibition sequence in step 1 includes any one of CDS characteristic sequence, miRNA, siRNA, sgRNA or antisense chain.

7. The use according to claim 6, characterized in that: The expression inhibition sequence is a CDS characteristic sequence, and the nucleotide sequence is shown in SEQ ID NO:

2.

8. The use according to claim 5, characterized in that: The original vector of the recombination inhibition vector in step 1 is any one of pTRV2, pFGC5941, pYLCRISPR / dCas9-KRAB, pRS300, and pBI121.

9. The use according to claim 8, characterized in that: The original vector of the recombination suppression vector is pTRV2.

10. The use according to claim 5, characterized in that: The step 2 is to introduce the recombination suppression vector obtained in the step 1 into the flower buds of the target plant.

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