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Rice leaf roller cadherin Cry toxin binding region coding gene as well as coding protein and application thereof

A technology of rice leaf roller and leaf roller calcium, which is applied in the directions of application, genetic engineering, plant genetic improvement and the like

Active Publication Date: 2021-09-17
JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no report about the cadherin gene of rice leaf roller, especially the related gene of toxin binding region

Method used

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  • Rice leaf roller cadherin Cry toxin binding region coding gene as well as coding protein and application thereof
  • Rice leaf roller cadherin Cry toxin binding region coding gene as well as coding protein and application thereof
  • Rice leaf roller cadherin Cry toxin binding region coding gene as well as coding protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Example 1 Amplification of the full-length sequence of the rice leaf roller cadherin gene

[0020] 1.1 Amplification of the conserved region sequence of the rice leaf roller cadherin gene

[0021] Rice leafroller was collected from Nanjing City, Jiangsu Province (118°52'E, 32°01'N). After indoor rearing for several generations, the 4th instar larvae of the same age were selected, RNA was extracted using Invitrogen Trizol kit, and the InvitrogenSuperScript TM II Reverse Transcriptase Kit reverse-transcribes total RNA to obtain cDNA template. The rice leafroller belongs to the Lepidoptera family Pidralidae, and four insects (O.nubilalis, O.furnacalis, D.saccharalis and C.pressalis) of the same family were analyzed using the bioinformatics software DNAMAN version 9.0. cadherin amino acid sequence alignment ( figure 1 ). According to the characteristics of the conserved regions of these four insect cadherins, a pair of degenerate primers (the upstream primer CmCad-con-F...

Embodiment 2

[0031] Example 2 Structure and evolution analysis of CmCad sequence of rice leaf roller cadherin

[0032] The secondary structure features of the rice leaf roller cadherin CmCad were analyzed using the ISRECProfile server (https: / / myhits.isb-sib.ch / cgi-bin / PFSCAN). The phylogenetic tree was constructed by the neighbor-joining method using MEGA 7.0 software, and the numbers at the nodes were the bootstrap values ​​of 1000 repetitions.

[0033] The predicted secondary structure of CmCad, consisting of a signal peptide (SIG), 11 cadherin repeats (CR), membrane-proximal extracellular domain (MPED), transmembrane domain (TMD), and cytoplasmic domain (CYT), is shown to be typical Cadherin structure of insect midgut ( figure 2 A).

[0034] To study the evolutionary relationship of cadherins from different insect species, a phylogenetic tree was constructed using the neighbor-joining method ( figure 2 B), the results show that 3 Nymphalidae, 3 Paleididae, 3 Papilionidae, 2 Silkwo...

Embodiment 3

[0035] Example 3 Construction, expression and purification of the Cry toxin-binding region of the rice leaf roller cadherin

[0036] The TBRs of cadherin Cry toxins in Lepidoptera are mainly located in the six CR domains (eg CR6-11 / CR7-12) and MPED near the cell membrane. Specifically, the CR6-MPED of the rice leaf roller has a total of 817 amino acids, and the truncated CmCad-CR6-MPED fragment was constructed, expressed and purified (J. Zhong, X. Hu, X. Zhang, Y. . Liu, C. Xu, C. Zhang, M. Lin, X. Liu. Broad specificity immunoassay for detection of Bacillusthuringiensis Cry toxins through engineering of a single chain variablefragment with mutagenesis and screening. International Journal of Biological Macromolecules, 2018, 107: 920 -928.). Specifically: use forward primer (CmCad-CR6-MPED-F whose nucleotide sequence is shown in SEQ ID No. 12) and reverse primer (nucleotide sequence as shown in SEQ ID No. 12) containing NcoI and NotI restriction sites respectively. CmCad-CR6-...

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Abstract

The invention discloses a rice leaf roller cadherin Cry toxin binding region coding gene with a nucleotide sequence as shown in SEQ ID NO.14 and a coding protein with an amino acid sequence as shown in SEQ ID NO.15 for the first time. A toxin binding region CmCad-CR6-MPED of rice leaf roller cadherin has a binding effect with Cry1Ac toxin, can be applied to prediction of rice leaf roller Cry toxin resistance. If the nucleotide sequence of the cadherin Cry toxin binding region gene of rice leaf roller to be detected is different from SEQ ID NO.14, a rice leaf roller receptor is subjected to gene mutation, the binding capacity is reduced, and the insect has certain resistance to Cry toxin. The coding protein can also be used for screening Bt protein aiming at the rice leaf folder; and if the Bt protein to be screened and a code have high affinity, the Bt protein to be screened is inevitably inserted into the midgut BBMV of the rice leaf folder, epithelial cell membrane is broken, and the Bt toxin insecticidal protein with high toxicity to the rice leaf folder can be rapidly screened or modified.

Description

technical field [0001] The invention belongs to the field of biological genetic engineering, in particular to a gene encoding the binding region of the cadherin toxin of the rice leaf roller and application thereof. Background technique [0002] The rice leaf roller Cnaphalocrocis medinalis is a major pest of rice in China and other Asian countries (Khan et al., 1988; Riley et al., 1995; Yang et al., 2018a). Rice leaf roller larvae feed by folding and defoliating rice leaves, causing severe yield losses in rice (Padmavathi et al., 2013; Senthil-Nathan, 2019). At present, chemical pesticides are mainly used for the control of rice leaf rollers. Due to environmental impact, pest resistance and other issues, chemical pesticides are not recommended for pests (Li et al., 2016; Xiao and Wu, 2019). Biopesticides are prepared from microorganisms or natural products and are harmless to the environment, and are effective alternatives to chemical pesticides (Chandleret al., 2011). Bt...

Claims

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Application Information

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IPC IPC(8): C12N15/12C12N15/70C07K14/435G01N33/68
CPCC07K14/43563C12N15/70G01N33/68
Inventor 仲建锋刘贤金徐重新高美静卢莉娜陈蔚谢雅晶张霄刘媛
Owner JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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