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Molecular Cloning and Expression Analysis of Na~+ Transport-related Genesand Construction of BIBAC Library of Phyllostachys Edilus

Author: YangYang
Tutor: LuoShuPing；ZhangZhiJun
School: Xinjiang Agricultural University
Course: Biochemistry and Molecular Biology
Keywords: phyllostachy edulis salt stress Na~+/H~+ antiporter gene clone genome library BIBAC
CLC: Q943.2
Type: Master's thesis
Year: 2010
Downloads: 54
Quote: 0
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Abstract


The pathway of maintain Na+ homeostasis in cytoplast is one of the major mechanisms of plant to tolerate salt. Isolation and research the genes which related to Na+ transport, is essentially for understanding the molecular basis of plant responded to salinity. In this study, the genes encode Na+/H+ antitporter and V-ATPase c subunit, was coloned from Moso bamboo. And expression pattern in different organs and tissues of these gene was analyzed under salt stress. The main result were as following:1) A novel vacuolar H+-ATPase c subunit gene (PeVHA-c) was cloned from Moso Bamboo. Sequence analysis showed that the PeVHA-c was 634 bp in length, including 495 bp of open reading frame(ORF), and encodes a protein of 165 amino acids with a predicted molecular mass of 16.6 kDa. PeVHA-c share high (>95%) homology with other vacuolar H+-ATPase c subunit from different species.2) A full-length cDNA encode vacuolar Na+/H+ antiporter protein named PeNHXl was isolated. This 2 290 bp sequence contained a 5’-UTR of 194 bp and a 3’-UTR of 447 bp, as well as an open read frame of 1 635 bp which encode a protein with 545 amino acid residues. The deduce transcripts PeNHX1 shared high homology with vacuolar Na+/H+ antiporters from other high plant. The data analysis showed that PeNHX1 have 12 conservative sequence of the transmembrane domain (TM) of which the 3rd TM have an amiloride-sensitive motif "LFFIYLLPPI".3) An cDNA for plasma Na+/H- antiporter genes named PeSOSl was isolated. This 3 674 bp sequence contained an completely open read frame of 3 417 bp and a 3’-UTR of 257 bp. The protein which encode by PeSOSI havel 138 amino acid residues. PeSOS1 protein had 12 predicted transmem-brane domains in the N-terminal region and a long cytoplasmic tail in the C-terminal portion. Phylogenetic analysis showed that PeSOSl and PeNHX1 was clustered in different groups which included plant plasma-type and vacuolar-type Na+/H+ antiporter respectively.4) Using the semi-RT-PCR technique to examine gene expression of PeNHX1、PeSOS1 and PeVHA-c in roots, stems, and leaves of seeding of Moso bamboo. The result showed that, under 200 mmol/L NaCl stress, PeVHA-c was strongly induced in all organs and tissues. The expression of PeNHXl and PeSOS1 were both enhanced in the roots, and the expression enhanced in leaves of PeNHX1 was also significant.Large genomic DNA fragments cloned in BIBAC vectors can be faithfully transmitted to transgenic progeny. So as to accelerate the procession of molecular breeding. In this study The method of obtaining HMW DNA, optimizing the condition of partially digestion and effected ligating and transforming system were studied, a BIBAC genome library construction system that suit our laboratory was established for the later work..

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