Construction And Sequence Analysis Of A SSH CDNA Library Of Sorghum Leaves Induced By Aphid

Sorghum[Sorghum bicolor (L.)Moench] is a staple crop in Africa and much of thedeveloping world because of its high-yield, drought, saline-alkali, superior tolerance of aridgrowth conditions. Sorghum is widely used as food, animal feed, biofuel, and for otheragricultural production, which has an important influence to the development of the nationaleconomy. The sorghum aphid [Melanaphis sacchari (Zehntner)] is a major pest of sorghum,because of its high reproductive rate, and spread rapidly, resulting in plant virus pandemic,directly to the sorghum causing serious harm, resulting in decline, lower quality, limiting theregion’s economic development. How effective sorghum aphid control has become an urgentneed to address the problem. Although the use of pesticides can effectively prevent a shorttime, but can easily make the aphid resistance is not conducive to long-term prevention andcontrol, and pesticide pollution of the environment, which contrary to the mainstream ofcurrent environmental protection, green agriculture.With the development of biotechnology,biological control with conventional breeding to cultivate high-yield, disease resistance andother new varieties with many good traits, has become a new research direction.In this study, we selected sorghum aphid resistant varieties of sorghum aphid infestation,"He nong16" and a sense of aphid species,"Qian san" as experiment materials. By SSHlibrary construction method, the sorghum aphid resistance gene was screened, analyzed thesedifferentially expressed aphid resistance gene sequences, we can know sorghum aphidresistance mechanism from gene level,and laid the theoretical basis of genetic disease forfuture breeding of sorghum aphid. The major findings are as follows:(1) Select sorghum aphid resistant species " He nong16" natural and artificial disease withaphids12h,24h,48h and72h treatment periods resistant leaves, a mixture of total RNA as thedriver side (Driver); Select sorghum sense of aphid species," Qian san "natural and artificialwith susceptible aphids12h,24h,48h and72h treatment periods of infected leaves, a mixtureof total RNA as the test side (Tester). Then we build a suppression subtractive hybridization(SSH) library. Library reorganization was83.5%, most of the insert PCR in length is200~650bp, about300bp on average. This indicated that SSH library with the aphid resistance ofsorghum has a high recombination rate and integrity.(2)240positive clones were randomly selected and sequenced. After removing vectorsequences and fragment assembly using Cross_match software Phrap program.The200 contigs were prediction and blast comparative analysis Blast Online by NCBI ORF Finderand online Blast Database. The results showed that the ORF sequences were highly relatedwith putative oxygen-evolving enhancer protein in chloroplast, adenylate cyclase-associatedprotein, ribosomal protein, putative senescence-associated protein. The contigs without ORFwere blasted and analyzed using online Blastx and Blastn and ESTs had highly homologousto genes sequences with known disease resistance in many plants, such as ribosomal protein,transport membrane protein, NBS-LRR disease resistance protein family-1, zinc fingerprotein, ATP synthase, NADH dehydrogenase, retrotransposon, Adenylyl cyclase-associatedprotein, and bZIP transcription factor etc.. These homologous genes involved in secondarymetabolism, energy metabolism, membrane transport, signal transduction and transcriptionregulation and so on. The candidate gene sequences compared with the known function than tothe homologous genes, involved in secondary metabolism, energy metabolism, cell signaling,disease prevention, such as protein synthesis in a variety of plant cell metabolism andresponse process.(3)" Henong16" respectively artificial disease with aphids12h,24h,48h and72h treatment,without aphids as contrast. Then we extracted RNA in each period reverse transcription RNAinto cDNA. Designed primers according to the sequencing of the EST gene sequences.Weverified SSH library by semi quantitative RT-PCR technology. The results show that, theexpression of these genes in the certain degree by aphid induced, but different expressionpatterns of gene, the expression of degree is different.The results showed that SSH technique is the feasibility study in sorghum aphidresistance mechanism, this establish basis for further cloning of sorghum aphid resistancegene.