Application and recombinant vector of bombyx mori nuclear polyhedrosis virus polygene inverted repeat sequence and bombyx mori lipase-1 gene

A technology of inverted repeat sequence and karyotype polyhedron is applied in the direction of introducing foreign genetic material using a vector, recombinant DNA technology, etc., which can solve the problems of limited improvement of resistance, consuming a lot of manpower and material resources, and no diapause period. The effect is obvious and the survival rate is improved

Active Publication Date: 2013-02-13
SOUTHWEST UNIVERSITY
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Problems solved by technology

However, the anti-virus strains prepared by using polymorphic silkworm varieties through transgenic interference have obvious defects in the following two aspects: ①The species used for production are generally diphelic diapause species, and the anti-virus strains prepared with polymorphic silkworm varieties It is not conducive to production promotion, and compared with diapause silkworm varieties, polymorphism silkworm varieties do not have a diapause period, and the maintenance of their subcultures can only rely on continuous breeding and breeding, which requires a lot of manpower and material resources to obtain. The maintenance and subculture of transgenic silkworm strains also greatly increases the risk of resource loss of obtained transgenic silkworm varieties
②The transgenic interference strain can only inhibit the replication and proliferation of the virus after virus invasion, and cannot play a role in other stages of virus infection
Bmlipase-1 is specifically expressed in the silkworm midgut, and then secreted into the silkworm intestinal juice to play a role. This protein can inhibit the inactivation of BmNPV virus in the intestinal juice, reduce the number of ODV viruses that invade the silkworm midgut cells, and thus improve the resistance of silkworm to BmNPV , but the resistance increase is limited

Method used

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  • Application and recombinant vector of bombyx mori nuclear polyhedrosis virus polygene inverted repeat sequence and bombyx mori lipase-1 gene
  • Application and recombinant vector of bombyx mori nuclear polyhedrosis virus polygene inverted repeat sequence and bombyx mori lipase-1 gene
  • Application and recombinant vector of bombyx mori nuclear polyhedrosis virus polygene inverted repeat sequence and bombyx mori lipase-1 gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 , Construction of transgenic vectors

[0031] 1. Construction of L4440-IE1P-FS-A3intron-FA-SV40 vector

[0032] (1) According to the reported BmNPV genome sequence and the silkworm nuclear polyhedrosis virus ie1 (GenBank: N0.X58442.1), gp64 (GenBank: N0. AB253773.1), lef1 (GenBank: N0. L09723.1), lef2 (GenBank: AF036245.1) and dna Polymerase gene (GenBank: No. D16231.1) is the sequence of 5 genes, artificially synthesized tandem DNA double-stranded sequence including partial sequences of the above 5 genes, named FS, as shown in SEQ ID NO.1. Set at the 5' end of FS with EcoRI Restriction site, the 3' end of FS is provided with Bam H Ⅰ Restriction site. Design and amplify the reverse complementary sequence FA of FS according to the artificially synthesized FS sequence, and the upstream primer is FA sense: 5'-ccc aagctt cgctgtctgaactatcacaa-3' (SEQ ID NO.2), underlined Hind Ⅲ Restriction site; downstream primer is FA anti: 5'-gc tctaga ata...

Embodiment 2

[0043] Example 2 , Transgene microinjection and screening of positive individuals

[0044] (1) Soak the silkworm eggs of the silkworm "Furong" in acid (the concentration of hydrochloric acid is 1.073 g / mL, the temperature is 46°C, and the time is 5 minutes) to relieve diapause, and then put them in a dark environment at 15°C and 80% humidity to catalyze Green for about 30 days until hatching, collect the ant silkworms and place them in a standard environment for rearing (temperature: 25°C, humidity: 80%). After the moths turn into moths, the male and female silkworm moths are mated for 4 hours. Diapaused silkworm eggs were used for microinjection in the next step.

[0045] (2) Arrange the laid silkworm eggs neatly on a clean glass slide, inject pb-HFPL recombinant vector and auxiliary plasmid A3H into 387 silkworm eggs with Eppendorf microinjector 2 hours after laying silkworm eggs, After sealing with non-toxic glue, put it in an environment of 25°C and 80% relative humid...

Embodiment 3

[0047] Embodiment 3, the resistance detection of transgenic system

[0048] Transgenic silkworms SW-H1, SW-H2, SW-H3 and non-transgenic silkworm SW (parental variety Hibiscus) were taken, and 5×10 5 Polyhedron / head and single head fed BmNPV virus by mouth, 3 replicated areas were set up for each of the 4 systems, 60 silkworms in each replicated area, and 3 non-challenge control areas SW(C) were set up at the same time, and the deaths were counted for 10 consecutive days Rate. Statistical results such as figure 2 shown by figure 2 It can be seen that the survival rates of SW-H1, SW-H2, SW-H3 and SW were 64.03%, 61.42%, 60.10% and 12.92%, respectively, the resistance of the transgenic system was significantly higher than that of the non-transgenic control, and the survival rate of SW-H1 An increase of 51.11%.

[0049] Compared with the results of Kanginakudru, S. in 2007 and Liang Jiang in 2012, it was found that simultaneously increasing the expression of resistance gen...

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Abstract

The invention discloses an application of a bombyx mori nuclear polyhedrosis virus (BmNPV) polygene inverted repeat sequence and a bombyx mori lipase-1 gene and specifically an application of the BmNPV polygene inverted repeat sequence and the bombyx mori lipase-1 gene to the construction of a recombinant vector; the vector can express a hairpin RNA (Ribonucleic Acid) for interfering with BmNPV multiple genes and bombyx mori lipase-1; BmNPV is inactivated by the incremental expression of bmlipase-1 (bombyx mori lipase-1) in the intestinal juice of a bombyx mori, and the number of BmNPV which invades intestinal cells of the bombyx mori is reduced; the expressed hairpin RNA can degrade the mRNA (Messenger Ribonucleic Acid) of a BmNPV gene and inhibit the replication and the proliferation of the virus in the Bombyx mori; and after the vector is used for producing a transgenic bombyx mori, the proliferation of BmNPV in the transgenicbombyx mor can be effectively inhibited, and the virus content is reduced, so that the survival rate of the bombyx mori infected with BmNPV is greatly increased.

Description

[0001] technical field [0002] The invention relates to the field of biotechnology, in particular to the application of the polyhedrosis virus polyhedrosis virus multigene inverted repeat sequence and silkworm lipase 1 gene, and also relates to the polyhedrosis virus polyhedrosis virus multigene inverted repeat sequence and silkworm lipase 1 Gene recombinant vector and preparation method. Background technique [0003] Bombyx mori is an important economic insect and a model organism of Lepidoptera. It has been bred and domesticated for more than 5,000 years in my country. Sericulture is one of the main sources of income for farmers in many regions and has made important contributions to the world's economic, cultural and social development. However, when silkworms are attacked by viruses, it usually causes huge losses to the silk industry. According to statistics, the losses caused by viral diseases account for about 70-80% of the total losses of silkworm diseases in seve...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/66
Inventor 蒋亮夏庆友程廷才赵萍王根洪
Owner SOUTHWEST UNIVERSITY
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