ASGNA Arabidopsis Identification, Insect Resistance Analysis And Genetic Transformation Of Cotton

Cotton is a very important economic crop in the world especially in the textile industry. However, cotton particularly easy to be infected by pests biting and phagocytosis infestation, so the cultivation of cotton has attracted wide attention in many countries. At present, transgenic insect-resistant cotton is mainly aimed at bollworm. But in recent years, sap-sucking insects, such as:aphids (Aphis gossypii), jassids (Amrasca devastans), whitefly (Bemisia tabaci), blind stinkbug (Pantatomidae), have become another major kind of pests following bollworm damage. As a result, cultivating anti-sap-sucking pests’ cotton is very important, and genetic engineering techniques also provide an effective means for the development of cultivating new cotton. In this research, we used a new insect-resistant gene, ASGNA, which was artificial syntheticed gene by our group, to detect its insect resistance using transgenic Arabidopsis and transient expression technology. And then we transfered the gene into cotton by genetic engineering technique in order to cultivating new anti-aphid transgenic cotton. The main results as follows:1) Kana resistance screening system was used to obtain pure seeds of five strains of Arabidopsis that transfected ASGNA; the result of detecting the level of transcription indicates that the five transgenic lines all have expression of ASGNA.By detecting the expression of the transgenic Arabidopsis’ roots, stems, leaves and flowers, the average amount of protein expression in fresh weight is 528.29-675.84ng/100g.2) The analysis of insect resistance of the five Arabidopsis lines transfected ASGNA showed that transgenic plants affect survival, birth and growth of aphids. Compared with the control group, the survival rate of aphids on transgenic Arabidopsis dropped 55.0%-80.7%, farrowing rate decreased 67.5%-82.5%; aphid body width reduces 0.20-0.36mm, aphid escaped numbers were significantly higher than the wild-type Arabidopsis. These results show that ASGNA has good resistance to aphids.3) The analysis of insect resistance of the five Arabidopsis lines transfected ASGNA against Plutella xylostella showed that:compared with the control group, the survival rate of Plutella xylostella on transgenic Arabidopsis had no obvious difference, but the Plutella xylostella growed on transgenic Arabidopsis DBM average weight reduced 8.84 mg each; Cocoon rate lower than the control group; we also observed that plutella xylostella which growed on the transgenic Arabidopsis had directly pupa with few spinning.It showed that ASGNA had certain resistance to chew type pests.4) The transient expression vectors,35s-ASGNA-eGFP-pzp211 and 35s-ASGNA-pART27, transferred into Agrobacterium tumefaciens LBA4404 respectively; then we use the cotton cotyledon transient expression system for expression of fuse protein. The analysis of detection of the expression of RNA levels and observation of green fluorescence, the cotton cotyledon transient expression system was used to express ASGNA, the result of detecting the level of transcription indicated all have expression of ASGNA. They showed that the cotton cotyledons transient expression system can be used in ASGNA gene expression at the transcriptional level and protein level.5) Using transient expression system to analyse insect resistance, we use the same grow period wild-type cotton cotyledons to feed Aphis gossypii as a control group, which transferred ASGNA-eGFP and ASGNA protein was experimental group. Compared with the control, aphids survival rate was 45.0%-52.5%, farrowing rate decreased 68.5%-82.5%, and the number of aphids escape was higher than control group. The data of above experiments indicate that transient expressing ASGNA has good resistance to aphidsin cotton cotyledon.6) The expression vector,35S-ASGNA- pART27, by using Agrobacterium to infect cotton hypocotyl callus regeneration, was transfected ASGNA into cotton cells. Through tissue culture, Kana resistance screening, several times’subculture after incubation, we ascertained the To transgenic cotton was transgenic plant by the way of the molecular identification.