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Gene GmAP1 capable of improving disease resistance of plants and application of gene GmAP1

A technology of disease resistance and genetics, applied in the fields of plant molecular biology and plant genetic engineering, can solve the problems of unclear resistance to Phytophthora and other diseases

Active Publication Date: 2019-04-19
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But its role in disease resistance against Phytophthora in plants remains unclear

Method used

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  • Gene GmAP1 capable of improving disease resistance of plants and application of gene GmAP1
  • Gene GmAP1 capable of improving disease resistance of plants and application of gene GmAP1
  • Gene GmAP1 capable of improving disease resistance of plants and application of gene GmAP1

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1. Cloning and sequence structure analysis of GmAP1 gene

[0055] Soybean (Glycinemax) seeds were directly planted in pots with moist vermiculite, cultivated in a greenhouse (25°C, 14h light / 10h dark), and the 1-week-old plants were used for RNA extraction.

[0056] Extraction of total RNA: Using soybean hypocotyls as the material, the total RNA was extracted using the RNA extraction kit from Omega Company in accordance with the instructions, and the RNA content and quality were detected with a spectrophotometer.

[0057] Reverse transcription to generate the first strand: take 0.7 μg RNA as a template, perform cDNA synthesis according to the instructions of Takara’s PrimeScript reverse transcriptase reagent kit, and dilute to 20uL for reaction. Take an appropriate amount of reverse transcription product for subsequent gene cloning PCR.

[0058] Use the first strand of cDNA as an RT-PCR template, and perform PCR in a conventional method to amplify the GmAP1 gen...

Embodiment 2

[0065] Example 2. Construction of the silencing vector pFGC::GmAP1:

[0066] Total soybean RNA was extracted, and cDNA was synthesized by reverse transcription. According to the sequence of GmAP1 cDNA, primers were designed to amplify some fragments for the construction of GmAP1 gene silencing vector.

[0067] pFGC-AscI-GmAP1-1F upstream primer: SEQ ID NO.9

[0068] 5'-ttacaattaccatggggcgcgccAATGAGCTTTGTGAAAAATT-3'

[0069] pFGC-AscI-GmAP1-2R downstream primer: SEQ ID NO.10

[0070] 5'-ttaaatcatcgattgggcgcgccTGCTGCCTCAGCAAATCCGA-3'

[0071] pFGC-BamHI-GmAP1-3R upstream primer: SEQ ID NO.11

[0072] 5'-aatttgcaggtatttggatccTGCTGCCTCAGCAAATCCGA-3'

[0073] pFGC-BamHI-GmAP1-4F downstream primer: SEQ ID NO.12

[0074] 5'-ctctagactcacctagatccAATGAGCTTTGTGAAAAATT-3'

[0075]The fragments of GmAP1 were amplified with the primers of SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, and SEQ ID NO.12, respectively. Perform PCR amplification of a partial gene sequence with a length of 297...

Embodiment 3

[0076] Example 3. Overexpression and silencing of the GmAP1 gene in soybean

[0077] Refer to the soybean hairy root transformation method established by Kereszt et al. (Kereszt et al., 2007).

[0078] Kereszt, A., Li, D., Indrasumunar, A., Nguyen, C.D., Nontachaiyapoom, S., Kinkema, M., and Gresshoff, P.M. (2007). Agrobacterium rhizogenes-mediated transformation of soybean to study root biology. Nature Protocols 2,948-952.

[0079] The specific operation steps are as follows:

[0080] 1) Sow soybean seeds (susceptible varieties such as Hefeng 47) in moist vermiculite, place them in a greenhouse (25°C, 16h light / 8h dark) for cultivation, after about 7 days, remove the watercress for sterilization in the experiment. At this time, the cotyledon differentiation ability is strong.

[0081] 2) After 6 days of planting beans, pick a single colony of Agrobacterium K599 into liquid LB containing antibiotics (50ug / mL kanamycin and 50ug / mL streptomycin), and culture at 28°C for 24h....

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Abstract

The invention provides a gene GmAP1 capable of improving the disease resistance of plants and application of the gene GmAP1, belongs to the field of plant molecular biology and plant genetic engineering, and relates to a plant source disease-resistant gene GmAP1 and a recombinant expression vector and application thereof. The gene is derived from a soybean, has the nucleotide sequence shown in SEQID NO.1, or has 70% or above of homology with SEQ ID NO.1. The invention further provides a gene silencing and recombinant expression vector. The gene is over-expressed to significantly promote the resistance of the soybean to phytophthora sojae and promote the resistance of tobacco to phytophthora capsici, and is an ideal gene capable of enhancing the disease resistance of the plants. Through agenetic transformation method, the gene is expressed in the soybean or tobacco, so that the gene obtains the resistance ability to various pathogenic bacteria, thereby being capable of improving the disease resistance of crops in the field. The gene GmAP1 can be used in the improvement of the disease resistance of crop breeding.

Description

technical field [0001] The invention belongs to the field of plant molecular biology and plant genetic engineering, in particular, the invention relates to a gene for improving plant disease resistance and its application. Background technique [0002] The crop disease caused by Phytophthora was once called "plant plague". Its onset is fast, the damage is serious, and there is a lack of effective fungicides. It is still a serious threat to global food production security. Among them, soybean Phytophthora sojae caused by Phytophthora sojae is a devastating disease in soybean production, causing losses of 1-2 billion US dollars in the world every year [1] . Potato late blight caused by Phytophthora infestans is an important disease in potato production, causing economic losses of up to US$ 3 billion annually worldwide [2] . Phytophthora soybean root rot was first introduced into China in the late 1980s, and then spread rapidly in most parts of China, and became one of the m...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/57C12N9/50C12N15/82C12N1/21A01H5/00A01H6/54A01H6/82C12R1/01C12R1/19
CPCC12N9/63C12N15/8282
Inventor 王源超郭宝佃王燕
Owner NANJING AGRICULTURAL UNIVERSITY
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