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Application of beckmannia syzigachne CYP704B1 gene

A technology of CYP704B1 and sagegrass, applied in the field of genetic engineering, can solve problems such as the difficulty of sagegrass management

Active Publication Date: 2019-06-04
INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] Long-term continuous and unscientific application of herbicides can easily lead to the emergence of weed resistance. In some areas of my country, wheat and rapeseed have developed serious resistance to herbicides, and with the emergence of non-target resistance, the weed Governance is more difficult

Method used

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  • Application of beckmannia syzigachne CYP704B1 gene
  • Application of beckmannia syzigachne CYP704B1 gene
  • Application of beckmannia syzigachne CYP704B1 gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0074] Example 1 Acquisition of CYP704B1 gene

[0075] The test selects sensitive population WC10-4 and resistant population WC10-10 collected from Jiangsu Province.

[0076] 1. Transcriptome sequencing

[0077] Select 3 samples of sensitive and drug-resistant Smilax 24 hours after treatment with methylsulfuron-methyl and 3 samples without treatment, and send them to the company for transcriptome sequencing.

[0078] 2. Acquisition of CYP704B1 gene

[0079] According to the transcript sequencing results, differentially expressed genes were screened, and the differentially expressed genes involved in the jasmonic acid signaling pathway could be determined through significant enrichment of pathways, and gene information and gene sequences were obtained from the sequencing results.

[0080] The nucleotide sequence of the CYP704B1 gene of Smilax chinensis is shown in SEQ ID NO:1.

Embodiment 2

[0081] The extraction of embodiment 2 total RNA

[0082] The test was collected from sensitive population WC10-4 and drug-resistant population WC10-10 in Jiangsu Province.

[0083] 1. Extraction of total RNA

[0084] Select 3 samples of Sensitive and drug-resistant Smilax 24 hours after treatment with Mesosulfuron-methyl and 3 samples without treatment, and extract total RNA respectively according to the instructions of the kit to obtain the corresponding total RNA solution.

[0085] 2. RNA agarose gel electrophoresis

[0086] Take 3uL total RNA solution, use 1% agarose gel for electrophoresis detection, the voltage is 160V, the electrophoresis time is 20min, and use gel imaging to scan. Such as figure 1 As shown, channel 1 from the left is Maker, channels 2 and 3 are both WC10-4; channels 4 and 5 are both WC10-10, and two clearly distinguishable main bands (28S and 18S) can be seen in ultraviolet detection, Indicates that the RNA is intact without degradation.

Embodiment 3

[0087] Example 3 Screening of primers for detection of CYP704B1 gene expression in Smilax

[0088] 1. Reverse transcription (synthesis of cDNA first strand)

[0089] (1) Prepare the reverse transcription system shown in Table 1:

[0090]Table 1

[0091]

[0092] (2) Carry out the reverse transcription reaction on the PCR instrument according to the following conditions:

[0093] 15min at 42°C, 5s at 85°C, and keep warm at 12°C.

[0094] (3) Dilute the product of (2) 3 times and set aside.

[0095] 2. Primer Design

[0096] (1) Obtain the gene sequence of CYP704B1 in Smilax chinensis by transcriptome sequencing.

[0097] (2) Design fluorescent quantitative PCR primers.

[0098] In step (2), use Oligo 7.0 software and Primer5.0 software to design primers. The designed primers are screened for specific primers at NCBI, and 3 pairs of primers are obtained after screening. The primer sequences, amplified fragment lengths and annealing temperatures are shown in Table 2:

...

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Abstract

The invention provides an application of a beckmannia syzigachne CYP704B1 gene. The invention further provides a fluorescent quantitation PCR primer (SEQ ID NO:2-3) for detecting the expression quantity of the beckmannia syzigachne CYP704B1 gene and a method for detecting the expression quantity of the beckmannia syzigachne CYP704B1 gene established based on the primer. The method is used for judging the resistance of beckmannia syzigachne to sulfonylurea type (especially mesosulfuron) herbicides. The method has the advantages of being simple, convenient, high in sensitivity and high in specificity, and has important application prospects in detection and research of the non-target resistance of the beckmannia syzigachne on the mesosulfuron. According to the application disclosed by the invention, the relationship of the beckmannia syzigachne CYP704B1 gene and the resistance of the sulfonylurea type herbicides is revealed for the first time, whether the beckmannia syzigachne generatesmetabolic resistance or not on the herbicides can be identified through detecting the expression quantity of the CYP704B1 gene, and the beckmannia syzigachne CYP704B1 gene has guiding significance ontimely finding out resistance, guiding scientific medication of farmland and relieving the further spread of the drug resistance.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular, to the application of the CYP704B1 gene of Smilax chinensis. Background technique [0002] Long-term continuous and unscientific application of herbicides can easily lead to the emergence of weed resistance. In some areas of my country, wheat and rapeseed have developed serious resistance to herbicides, and with the emergence of non-target resistance, the weed Governance is more difficult. At present, studies have shown that cytochrome P450 can metabolize herbicides into non-toxic substances through dealkylation, ring methyl hydroxylation, hydroxylation of aromatic rings, desulfurization oxidation, and catalyzed ester bond cleavage. , and the increased expression of some genes in the P450 family will make the originally sensitive weed population resistant to herbicides (Yasuor H, Osuna MD, Ortiz A, et al. Mechanism of resistance to penoxsulam in late watergrass[Echinoch...

Claims

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Application Information

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IPC IPC(8): C12Q1/6895C12Q1/6851C12N15/29C12N15/11
Inventor 崔海兰李香菊王京京陈景超李政黄兆峰于惠林
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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