关键词: 杜仲, EuCPI, 基因克隆, 耐盐, 耐高温, 抗虫

Abstract:

Objective: The full length sequence of EuCPI gene was cloned from Eucommia ulmoides seed kernel and its structural characteristics were analyzed. The stress resistance function of EuCPI and its impact on the growth and development of Tenebrio molitor were explored. The results can provide a theoretical basis for further study of the stress-resistance mechanism in the future. Method: The RNA of E. ulmoides kernels was extracted and reverse transcribed into cDNA, the full-length cDNA sequence of EuCPI gene was amplified by RACE technology; the physicochemical properties and structural functions of the encoded protein were analyzed and predicted by using bioinformatics websites; the fragment containing EuCPI gene was inserted into pET28a to construct a prokaryotic expression recombinant vector, and the recombinant protein was investigated by SDS-PAGE, Western Blot, and protein purification tests; to preliminarily identify the effect of EuCPI gene on Escherichia coli, the differences in growth curves under salt and high temperature stress between E. coli containing EuCPI gene and control group were explored using recombinant prokaryotic expression system; in addition, the effect of EuCPI gene on the growth and development of T. molitor was studied by feeding T. molitor with the induced E. coli containing EuCPI gene. Result: The full-length sequence of EuCPI gene was 547 bp(GenBank accession number: MT702592), the gene contained a 309 bp open reading frame (ORF), encoding 102 amino acid residues. The predicted molecular weight of the putative protein was 11.17 kDa with the isoelectric point(pI) of 6.41. The protein was a stable and hydrophilic protein, the instability index was 27.73 and the total average hydrophilicity was -0.416. The amino acid sequence analysis showed that EuCPI protein had no transmembrane structure and no signal peptide, mainly composed of 50% α-helix, 36.27% random coil, 11.76% extended chain and 1.96% β-sheet, which belonged to the cysteine proteinase inhibitor family. Phylogenetic tree analysis showed that EuCPI protein had high homology with CPI of Quercus suber. The recombinant protein with the size of 15.29 kDa was induced and purified by the constructed prokaryotic expression vector pET28a-EuCPI. The growth curve of high temperature stress showed that the E. coli containing EuCPI gene was basically the same as that of the control group in the first 10 hours at 37℃, however the growth rate of the BL21(DE3) E. coli containing EuCPI gene was significantly greater than that of the control group in the first 10 hours at 42℃ and 50℃, and the control group showed significant growth inhibition. The salt stress test on solid medium showed that with the increase of NaCl concentration, both the test group and the control group showed growth inhibition, but the number of colonies containing EuCPI gene was significantly more than that of the control group, especially at 0.75 mol·L^-1, the former had more single colonies survived and the latter had no single colonies survived. Under 0.25 mol·L^-1 NaCl concentration in the first 8 hours, the growth curve of salt stress in liquid medium showed that the growth rate of the BL21(DE3) E. coli containing EuCPI was significantly faster than that of the control group displaying obvious growth inhibition. The forage tests showed that the body mass of T. molitor fed containing EuCPI gene decreased, while the body mass of the control group increased (P < 0.01). Conclusion: The EuCPI gene can improve E. coli tolerance to high temperature and salt stress, and the recombinant bacteria containing EuCPI gene can inhibit the growth and development of T. molitor

Key words: Eucommia ulmoides, EuCPI, gene cloning, salt resistance, high temperature resistance, insect resistance