Gene Cloning And Function Research Of CsSPI In Tea Plant And EgGL In Grey Tea Geometrid

Serine proteinase inhibitor(SPI)have been reported to play an important role in protecting plants from herbivore attack.However,little is known about their function in tea plants.The grey tea geometrid Ectropis grisescens(Lepidoptera: Geometridae),a close species of tea geometrid Ectropis oblique Prout(Lepidoptera: Geometridae)is one of the major pests of tea production.However,little is known about the resistance of different tea cultivars to E.grisescens.Phytophagous insect saliva elicitors play an important role in plant defense responses.Analysis the function of SPI gene CsSPI of tea plant in insect-resistance and the role of E.grisescens glucosidase in tea plant induced defense responses will provide scientific basis for tea resistance cultivar selective breeding and co-evolutionary relationship between E.grisescens and tea plant.We obtained SPI gene CsSPI of tea plant and E.grisescens glucosidase gene by gene clone technology and analysed the expression characteristics of these genes.We determined function of SPI gene in insect-resistance by means of studying the relationship between the activity of serine protease inhibitors in different tea cultivars and the insects growth.Using the technology of RNA interference,we constructed a L4440-EgGL recombinant expression vector.The results of the study are as follows:(1)A CsSPI gene has been cloned from tea plant using RT-PCR.It is 1669 bp in length containing a 1170 bp open reading frame(ORF).It encods a protein of 389 amino acids with a calculated molecular weight of 42.43 kD and a isoelectric point of 6.36.Comparision with other known SPI amino acid sequences showed that CsSPI contains the conserved sequences.Compared to other tissues,flower has the highest expression level of CsSPI in tea plant.In EB cultivar,the highest expression of the gene was detected in leaf and stem.(2)The growth and development of E.grisescens larvae and pupae raised on different tea cultivars showed that EB and ZS as resistant tea cultivars and BZY and LT as susceptible tea cultivars.And the activity of serine protease inhibitors in EB and ZS were significant higher than BZY and LT,suggesting that the expression of tea SPI may be related to its resistance to the moth.(3)An EgGL has been cloned from E.grisescens larvae using RT-PCR.It is 2717 bp in length containing a 2490 bp open reading frame(ORF).EgGL gene encoded a protein of 829 amino acids with a calculated molecular weight of 94.99 kD and a isoelectric point of 8.71.The amino acid of E.grisescens EgGL was in 98% similarity to that of E.oblique which is a close species of E.grisescens.(4)We have constructed a L4440-EgGL recombinant expression vector using gene recombinant technique and transformated it into E.coli HT115(DE3).