Development Of Anti-chilo Infuscatellus Sugarcane Germeplasm Based On Silencing The CiChi1 Gene By RNAi

Chilo infuscatellus（Snellen）that belongs to the family Lepidoptera is one of the main pests of sugarcane and become prevalent in most sugarcane growing areas in China.However,due to the lack of sugarcane resistant germplasm,it is difficult to cultivate high-efficiency and insect-resistant sugarcane varieties by conventional hybrid breeding.Long-term application of chemical agents will lead to a series of problems,such as insect resistance,pesticide residues and environmental pollution.It is urgent to find out an effective method to control the C.infuscatellus in sugarcane.Therefore,in this study,we carried out the full-length cloning,expression and functional analysis of chitinase-related genes of C.infuscatellus based on sequencing and analysis of the transcripts of C.infuscatellus at different growth stages.Then screening of RNAi target genes and their corresponding high-efficient dsRNA sequences for the control of C.infuscatellus insect pests was performed.Subsequently,the RNAi plant expression vector containing CiChi1 gene of sugarcane C.infuscatellus was introduced into the leaf roll tissues of sugarcane by particle bornbardment.A batch of transgenic plants with resistance to C.infuscatellus were generated.Our findings were listed as below for suppling with a new way for integrated pest management in sugarcane.（1）The transcriptome of C.infuscatellus was sequenced with different developmental stages,and a total number of 4424800002 nucleotides were obtained.These reads were assembled into 95921 unigenes with an average length of 1542 bp,and the corresponding N50 and N90 were2591 bp and 653 bp,respectively.These unigenes are annotated in different databases,and a total of 53815 unigenes can be annotated into at least one database,accounting for 56.1%.Of these,47849,36605,and19406 unigenes were successfully annotated in the Nr database,the Pfam database and the Nt database,accounting for 88.9%,68.0%and 36.1%of the total annotated unigenes,respectively.In addition,accounting for 68.5%of the total annotated unigenes,36890 unigenes were successfully annotated in 55 GO entries.（2）Through screening of C.infuscatellus transcriptome data,full length of three C.infuscatellus chitinase genes（CiChi1,CiChi2,CiChi11）and one chitinase domain-containing protein 1（CiChid1）were successfully cloned.Fluorescence quantitative PCR（qPCR）revealed that CiChi1 was highly expressed in adults,but and lower in 6^th instar.The CiChi2 was highly expressed at 6^th instar and adult,and had the lowest expression at 5^th instar.The expression level of CiChi11 was lowest in the adult stage and highest in the 3^rd instar;CiChid1 was highly expressed at5^th instar,but lowly expressed at 1-3 instar.The CiChi11 and CiChid1were highly expressed in the midgut tissues,but lowest expressed in the head and epidermal tissues.CiChi1 and CiChi2 were highly expressed in epidermal tissue,but lower level in the head and midgut tissues.（3）Functional analysis of C.infuscatellus CiChi1 gene using RNAi technology showed that the injection of dsRNA targeted at CiChi1 gene affected the growth and development of C.infuscatellus.Compared with the control,the expression of CiChi1 gene decreased by 70-80%in the surviving borers and went down by 62%in the larvae mortality at 7 days post-injection.（4）The interfering sequence hairpin structure of the fragment was constructed into the plant expression vector pUC18 and resulted to the pUC18-CiChi1 vector.Then,with this plasmid together with the pTEM73vector（carring the bar gene the herbicide resistance to glyphosate）were introduced into the leaf roll tissues of sugarcane by the gene gun-mediated method.The vector containing the EGFP sequence was a negative control.79 transgenic plants with dsDNA of CiChi1 sugarcane and 5 transgenic plants with EGFP were identified by screening with 18%glufosinate and PCR detection.qPCR analysis showed that CiChi1-transgenic plants could express interference fragments,but the expression levels were different in divese transgenic plants.