Studies On Biochemical Mechanism Of Resistance To Aphid And Plant-mediated RNAi To Control Aphids

Wheat aphid, an important pest in the wheat planting areas of China, can cause considerable damages to wheat production and quality by sucking plant juice, excreting of honeydew, and spreading virus, respectively. With the changes of irrigation conditions and agricultural ecological environment, the wheat production and quality were easily affected by aphids in recent years. The Chemical pesticides are now used to control the wheat aphids, which may lead to many problems about human health, environment and pest resistance in using pesticides. In order to offere reference to crop breeding for aphid resistance, resistant varieties and lines to aphid were screened from150materials in2010/11and2011/12in this study, and then their morphological characters and physiological traits were studied. The transgenic wheat lines for aphid-resistance were developed by using plant-mediated RNAi. The key results are as follows:1,150wheat accessions were evaluated to aphid at booting stage and grain-filling stage in field conditions in2010/11and2011/12in Shangzhuang experiment stations of china agricultural university. The results showed that6resistant varieties/lines,08P20, KOK1679, Heng6632, Ji38, Lingkang15and Luomai4showed resistant to aphids.43wheat varieties/lines with different degrees of aphid resistance were studied by55SSR markers to explore the genetic diversity. Fifty-five markers were screened and a total of370allelic variants were detected in43wheat varieties, ranging from2to18alleles per marker with an average value of6.7. The PIC (polymorphism information contents) for each polymorphic primer varied from0.02-0.91, with an average value of0.65. The mean genetic distance (GD) among43genotypes was0.52, varying from0.3-0.90, and43varieties were clustered into five groups under the genetic distance.2, There were correlation between Plant height, spike length, spikelet number, flag leaf length, growing period and the ratio of aphid number, but there were not associated between peduncle length, awn length, spikelet density and ratio of aphid numbers. The physiological responses of6wheat genotypes to Sitobion avense (Hemiptera:Aphidoidea) were examined in grain-filling stage. Soluble sugar content, protein content and chlorophyll content of6cultivars showed a general trend of reduction after aphid-infestation compared to their respective controls. However, it showed that there were relatively higher content of free proline, total phenols and phenylalanine ammonialyase activity after aphid-infestation, and the susceptible cultivars had greater increases compared to resistant cultivars.3, Three fragment of Carboxylesterase gene (CbE E4), Lipoprotein lipase gene (LPL) and Gqa gene were cloned by using specific primers. The fragment of CbE E4gene was sequenced, and its nucleotide sequence homology with Myzus persicae was83.1%. The fragment of LPL gene were sequenced and the nucleotide sequence homology with pea aphid (GenBank:XM-0019507372, XM-001944358.2, FF314537) LPL were86.79%,75.37%,78.86%, respectively. The cDNA sequences of Gqa fragment showed98.1%similarity to previous study (GenBank:EF638906). There was continuous higher expression in different development of aphid, but LPL and Gqa gene showed no significantly expression. Those3genes could be up-regulated by phoxim solution.The RNAi vectors were transferred to embryo callus of wheat by particle bombardment. Bioassays were performed by applying neonate aphids to homozygous transgenic plants in the T2and T3generation. Results revealed that continuous feeding of transgenic diet reduced CbE E4mRNA level in the fed S. avenae and inhibited CbE E4enzyme activity and insect reproduction. LPL mRNA level in the fed aphids was reduced of7.6%and11.4%afer feeding transgenic plants of dsLPL-1and dsLPL-4for five days, and the transgenic plant of dsLPL-1can significantly inhibited S. avenae reproduction, but the aphid reproduction was not affect by dsLPL-4. The Gqa mRNA level were respectively reduced of6.8%,11.4%and18.7%for1days,3days and7days; the reproduction of aphid was inhibited of21%and26%for11and15days on transgenic line of dsGqa6-4.