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A brown planthopper gene nl19243 and its encoded product and application

A brown planthopper and transgenic plant technology, applied in the application, genetic engineering, plant gene improvement and other directions, can solve the problems of no brown planthopper cloned, reduce related genes, and the lethal effect of brown planthopper is not obvious, and achieve the promotion of rice planthopper resistance breeding, good The effect of anti-insect effect

Inactive Publication Date: 2015-09-30
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the use of these transgenic rice lines can reduce the transcription level of related genes to a certain extent, the lethal effect on BPH is not obvious
Therefore, so far, no relevant target gene of brown planthopper has been cloned at home and abroad, and the purpose of controlling brown planthopper can be achieved by silencing the gene through transgenic rice

Method used

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  • A brown planthopper gene nl19243 and its encoded product and application
  • A brown planthopper gene nl19243 and its encoded product and application
  • A brown planthopper gene nl19243 and its encoded product and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Embodiment 1, Nl19243 Gene acquisition and sequence analysis

[0030] 1) Extraction, quality inspection and first-strand synthesis of total cDNA from salivary gland RNA of brown planthopper;

[0031] 2) Synthesized by polymerase chain reaction (PCR) from the first strand of total cDNA Nl19243 CDS Fragment

[0032] Upstream primer (SEQ ID No.3): 5'- GTCCCGTTCACGACACTA -3'

[0033] Downstream primer (SEQ ID No.4): 5'- TCTGATATTTCAACTGCCTC -3'

[0034] PCR amplification conditions: 98°C×3min→(98°C×30sec→55°C×30sec→72°C×50sec)×30 cycles→72°C×10min, see attached figure 1 .

[0035] 3) pMD19- Nl19243 Cloning vector construction and gene base analysis

[0036] The PCR product was cloned into the pMD19-T vector of Takara Company to obtain pMD19- Nl19243 vector, and transformed into TG1 Escherichia coli by heat shock method, will contain pMD19- Nl19243 The carrier TG1 bacterial liquid was sent to Nanjing GenScript Company for sequencing. Sequence alignment analysis w...

Embodiment 2

[0037] Embodiment 2, brown planthopper Nl19243 dsRNA synthesis and injection RNAi effect

[0038] 1) From the above pMD19- Nl19243 Obtained by polymerase chain reaction (PCR) in the cloning vector Nl19243 The middle 425bp fragment, and continue to synthesize Nl19243 dsRNA fragments. At the same time with GFP Plasmid as template, obtained by PCR GFP The middle 860bp fragment, and continue to synthesize GFP dsRNA fragment

[0039] ds Nl19243 -T7-F (SEQ ID No. 5):

[0040] 5’- GGATCCTAATACGACTCACTATAGG TGGCTCACTCGTACTTCTT-3’

[0041] ds Nl19243 -T7-R (SEQ ID No. 6):

[0042] 5'- GGATCCTAATACGACTCACTATAGG CCACTTCACTCCGTCCTG -3'

[0043] ds GFP -T7-F (SEQ ID No. 7):

[0044] 5'-GGATCCTAATACGACTCACTATAGGAAGGGCGAGGAGCTGTTCACCG-3'

[0045] ds GFP- T7-R (SEQ ID No. 8):

[0046] 5'-GGATCCTAATACGACTCACTATAGGCAGCAGGACCATGTGATCGCGC-3'

[0047] PCR amplification conditions:

[0048] Nl19243 : 98℃×3min→(98℃×30sec→62℃×30sec→72℃×30sec)×30 cycles→72℃×10min

[0049] ...

Embodiment 3

[0062] Embodiment 3, transgene Nl19243 Studies on the function of dsRNA against insects in rice

[0063] 1. Design forward and reverse primers, PCR amplify the 425bp long 155th to 579th Nl19243 genetic DNA fragment.

[0064] 2. DNA subcloning Nl19243 The gene fragment is inserted into the pMECE vector forward and reverse at the same time, and the forward and reverse connections are obtained. Nl19243 Gene fragment RNAi expression vector pMECE- Nl19243 ( Figure 4 ).

[0065] 3. Add pMECE- Nl19243 , into Agrobacterium EHA105 by electric shock method to obtain Agrobacterium engineered cell line. The rice callus was infected by Agrobacterium transfection method, and the co-infected rice callus was cultured on NBDS medium containing hygromycin, and the first selection was 20 days. After the new resistant callus grows, the resistant callus is stripped from the parent body and transferred to a new selection medium NBDS2, and one resistant callus is a strain. After the resis...

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Abstract

The invention discloses a brown planthopper gene Nl19243 as well as an encoding product and application thereof. The gene Nl19243 has the DNA sequence shown in SEQ ID No. 1. The DNA sequence is composed of 787 nucleotide bases and contains a complete encoding frame composed of 654 nucleotides, and the coded proteins contains a small-molecular protein with 217 amino acid residues. Research finds that the gene is related to the survival of brown planthopper. An RNAi vector for the Nl19243 is constructed and then is transferred into rice to express dsRNA of the gene Nl19243 in a transgenic rice body, so as to achieve the purpose of improving the resistance of rice to brown planthopper. The brown planthopper gene Nl19243 can be widely applied to crop breeding, particularly breeding of insect-resistant rice.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a brown planthopper gene Nl19243 And its coding products and applications. Background technique [0002] Rice ( Oryza sativa L.) is one of the most important food crops in China and even in the world. In China, the annual rice planting area is about 30 million hectares 2 , accounting for nearly 1 / 2 of the planting area of ​​grain crops, and rice production accounts for 45% of the total grain output. In recent years, China's rice planting area has declined, but rice production still accounts for about 40% of the total grain output. Rice production is directly related to China's food security. However, the annual loss of rice production due to rice plant diseases and insect pests in the whole country is huge, although the control still causes economic losses of 4 million to 5 million tons, and even the grains are not harvested in severe disaster years. Therefore, how to increase ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/12C07K14/435A01H5/00
Inventor 娄永根陈红丹纪锐李恒
Owner ZHEJIANG UNIV
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