Cloning And Expression Analysis Of Stress Tolerance Genes LcPIP And LcLHP In Leymus Chinensis

Leymus chinensis is widely distributed in natural saline-alkali soil in northern China depended on its strong tolerance ability. It has accumulated resistance genes in the evolutionary process. It is a good test material of plant stress mechanism and the resistance genes separation. Based on the PIP EST of L. chinensis, A complete cDNA of LcPIP and LcLHP from200mM of Na2CO3stressed L. chinensis were cloned using the method of the RACE and RT-PCR technology. The sequences of LcPIP and LcLHP genes were predicted and analysised by bioinformatics method. Expression patterns were analysised under different salinity stress treatment by semi-quantitative RT-PCR. The prokaryotic expression system was used to inducible expression of GST fusion proteins. The test results will lay the foundation for the study of the stress mechanism and resistance function of L. chinensis gene from the molecular level.(1) The cDNA of LcPIP gene from salinity-alkalinity stressed L. chinensis were cloned using the method of the RACE and nested PCR. The sequences and bioinformatic prediction analysis indicated that LcPIP contained a879bp open reading frame encoding292amino acids. The NPA boxes and MIP family of characteristic sequence "HINPAVTFG" were contained in LcPIP. LcPIP was highly homologous with the reported source of monocots PIP1family proteins. PIP shared highly conserved sequences in the transmembrane domains with the PIP family proteins from wheat, barley and corn. The result indicated that such proteins had higher conservative in the process of genetic evolution and LcPIP protein belongd to the aquaporin PIP1family.(2)The cDNA of LcLHP gene from salinity-alkalinity stressed L. chinensis were cloned using the method of the RACE and nested PCR. The sequences and bioinformatic prediction analysis indicated that LcLHP cDNA contained an open reading frame of537bp encoding178amino acids. LcLHP contained PLAT/LH2domain. The homology analysis showed that LcLHP was highly identified with Arabidopsis Lipase/lipooxygenase and PLAT-plant-stress domain-containing protein. The N-terminal signal peptide and enzyme function were included Comprehensive prediction results concluded that the gene of L. chinensis was Lipoxygenase homology protein gene and named LcLHP.(3) Semi-quantitative RT-PCR showed that the expression of LcPIP and LcLHP were regarded as a background level in the non-stressed leaf. The gene expression showed a single peak trend under200mM Na2CO3stress at different times. The results showed that the gene of LcPIP was up-regulated gradually until12h, and the LcPIP expression level of peak was24h. The recombinant expression vector pGEX-LcLHP was constructed. IPTG was used to induce pGEX-LcLHP to express GST-LcLHP in Escherichia coli. The fusion protein was validated by Western blot using GST antibody. The LcPIP gene was cloned from pGMT-LcPIP plasmid, and constructed it into expression vector pGEX-KG. The pGEX-LcPIP recombinant expression plasmid was confirmed by using double digestion and PCR. But the fusion protein was not detected in prokaryotic expression system under different induction conditions.