Molecular Cloning,Functional Identification And Expression Characteristic Of The HPL Gene From Camillia Sinesis Induced By Ectropis Oblique Feeding

C6-Volatile is one of the key material which is indirect defense against pests in the plants, fatty acid hydroperoxide lyase is a key enzyme in its synthesis in plants. However, it hasn’t been reported that HPL gene of tea and the relationship between HPL gene and insect feeding. The EST fragment of hydroperoxide lyase (HPL) was screened form SSH library of tea induced by Ectropis oblique feeding. On this basis, research molecular cloning, functional identification and expression characteristic of the HPL gene from Camillia sinesis induced by Ectropis oblique feeding. The main results are as follows:(1) A full-length cDNA of HPL was first obtained by3’/5’-RACE (rapid amplification of cDNA ends) techniques from fresh leaves of Tea plant. The sequence length is1662bp. There are five open reading frame from the initiation codon. The longest ORF encode a polypeptide of491amino acid residues and named as CsHPL(GenBank：HM440156), theoretical molecular mass and PI of54.88kD and8.02, respective. The results showed that the sequence presented71%similarity with the guava13-HPL genes by multiple sequence alignment analysis. CsHPL contains four highly conserved domains (domainA, B, C and D) of Cytochrome P450family by conserved domain analysis. Its domain A (I helix) is isoleucine instead of conserved threonine. Its domain D has some specific sequences NKQAA in the vicinity of highly conserved cysteine residues. Phylogenetic analysis showed that CsHPL and guava13-HPL together are CYP74B subfamily.(2) Prokaryotic expression Analysis of the CsHPL gene resulted in the production of a special fusion protein which found in supernatant and inclusion body (MW: about60kD). In vitro enzymatic reactionshowed that CsHPL of prokaryotic expression has activity when use13-HPOT as substrate and hasn’t activity when use9-HPOT as substrate. Therefore, CsHPL is classified13-HPL. CsHPL of prokaryotic expression optimum pH is7.0, optimum temperature is25℃. Prokaryotic expression of five different ORF of CsHPL gene, the results showed that the all special fusion protein has activity and significant about their active change.(3) Volatile substances of products was analyzed by GC-MS, identified as (z)-3-hexenal when use13-HPOT as substrate.(4) Southern blot analysis, indicate that CsHPL is present as single copy in Tea plant genome.(5) qRT-PCR analysis showed that two varieties of Longjing43and Shuchazao induced by Ectropis oblique feeding, the expression level of CsHPL gene can significantly increase on3-9h, and attain maximum expression in this period of time. However, the time of maximum expression is difference between Longjing43and Shuchazao. After Longjing43induced by Ectropis oblique feeding, the expression level of CsHPL gene increased to maximum on3h which is13.6times compared with the control. However, the expression level of CsHPL gene increased to maximum on9h that is3.9times compared with the control. Mechanical damage of two varieties, the expression of CsHPL gene showed a similar rule.