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Application of rice potassium ion transport protein gene OsHAK9 in improving seed germination capacity under salt stress

A technology for rice seeds and transporters, which can be used in applications, genetic engineering, plant genetic improvement, etc., and can solve problems such as fewer clones

Active Publication Date: 2020-10-30
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, under salt stress, seed germination is affected by both osmotic and ionic effects
So far, few genes related to seed germination tolerance to salt stress have been cloned, and further exploration and cloning are needed

Method used

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  • Application of rice potassium ion transport protein gene OsHAK9 in improving seed germination capacity under salt stress
  • Application of rice potassium ion transport protein gene OsHAK9 in improving seed germination capacity under salt stress
  • Application of rice potassium ion transport protein gene OsHAK9 in improving seed germination capacity under salt stress

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1: Gene Mapping

[0043] (1) Materials and methods

[0044] 1. Materials: The salt-sensitive mutant gss1 (attached figure 1 shown), and crossed with wild-type Nipponbare (WT) to construct F 2 The isolated population was used for the detection and localization of salt-tolerant loci during seed germination using the MutMap method based on the resistance-sensitivity pool (BSA). Depend on figure 1 A. figure 1 B. figure 1 C It can be seen that the seed germination rate, germination speed or seedling growth of the gss1 mutant seeds under salt stress is significantly lower than that of the control japonica Nipponbare seeds.

[0045] 2. Screening of individual plants in resistant and susceptible pools: in F 2 In the segregation population, 150 progeny seeds of individual plants were randomly selected for identification of seed germination under 200mM salt stress, and 20 individual plants of extreme salt resistance and salt sensitivity were selected according to th...

Embodiment 2

[0049] Embodiment 2: gene cloning

[0050] Using the cDNA of the japonica rice variety Nipponbare seedling leaves as a template, the sequence of the OsHAK9 gene was cloned by using touchdown PCR amplification technology. The sequence of the upstream primer is shown in SEQ ID NO.3 in the sequence table, and the sequence of the downstream primer is shown in SEQ ID NO.4 in the sequence table. Show. The nucleotide sequence and amino acid sequence of the rice OsHAK9 gene are obtained, the nucleotide sequence is shown in SEQ ID NO.1 in the sequence table, and the amino acid sequence is shown in SEQ ID NO.2.

[0051] OsHAK9-F1: 5'-GCTGATTCTCTCGATTGATTAC-3' (SEQ ID NO.3)

[0052] OsHAK9-R1: 5'-AGCAGAGAGATTTGATTTTGAA-3' (SEQ ID NO.4)

[0053] The PCR reaction system is:

[0054]

[0055] The reaction program is: pre-denaturation at 95°C for 5min, denaturation at 95°C for 30s, annealing at 60°C for 30s, extension at 72°C for 90s, 8 cycles, denaturation at 95°C for 30s, annealing a...

Embodiment 3

[0056] Example 3: Complementary transgenic plants

[0057] (1) Amplification of the promoter sequence: Using Nipponbare genomic DNA as a template, PCR amplification technology was used to amplify the sequence about 2.4kb upstream of the OsHAK9 initiation code. The upstream primer sequence is shown in the sequence table as SEQ ID NO.5, The downstream primer sequence is shown in SEQ ID NO.6 in the sequence table, and the OsHAK9 gene promoter sequence shown in SEQ ID NO.7 is obtained.

[0058] OsHAK9-F2: 5'-GCAACAGCAAAGGTCACTCT-3' (SEQ ID NO.5)

[0059] OsHAK9-R2: 5'-ACCAGTGGATCGGATAGTCC-3' (SEQ ID NO.6)

[0060] The PCR reaction system is:

[0061]

[0062]

[0063] The reaction program was: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30 s, annealing at 56°C for 30 s, extension at 72°C for 90 s, 30 cycles, complete extension at 72°C for 10 min, 32 cycles, and storage at 4°C.

[0064] (2) Construction of recombinant plasmids: Based on the principle of ov...

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PUM

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Abstract

The invention belongs to the field of seed science and technology and discloses application of a rice potassium ion transport protein gene OsHAK9 in improving seed germination capacity under salt stress. The nucleotide sequence of the rice potassium ion transport protein gene OsHAK9 is as shown in SEQ ID NO. 1, and a coded amino acid sequence is as shown in SEQ ID NO.2. According to the invention,the gene OsHAK9 is obtained by cloning from a rice salt sensitive mutant gss1 through a MutMap method, and functional complementation experiments prove that the OsHAK9 can improve the germination capacity of rice seeds under salt stress. The gene can be applied to high-activity seed rice variety breeding and genetic improvement of rice seed germination traits under salt stress and has important significance for direct seeding rice production.

Description

technical field [0001] The invention belongs to the field of seed science and technology, and relates to the application of the rice potassium ion transporter gene OsHAK9 in improving the germination ability of rice seeds under salt stress, and is specially used for the breeding of rice varieties with high vigor seeds and the genetic improvement of rice seed germination traits under salt stress . Background technique [0002] Rice (Oryza sativa L.) is an important food crop in my country, and about 30% of the rice arable land in the world is affected by salt damage. With the development of the economy, direct-seeding rice and machine-transplanted rice are becoming more and more common, and it is becoming increasingly important to improve the germination ability of rice seeds under salt stress conditions. [0003] Seed germination is a complex quantitative trait controlled by multiple genes. In the past ten years, QTL analysis has been used to locate multiple QTLs related t...

Claims

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Application Information

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IPC IPC(8): C07K14/415C12N15/29C12N15/82A01H5/10A01H6/46
CPCC07K14/415C12N15/8273
Inventor 程金平张红生曾鹏
Owner NANJING AGRICULTURAL UNIVERSITY
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