Primary Study on CLC1 Gene Identification and Its Function in Glycine Max, Salt-born Glycine Soja and Their Hybrid
|School||Nanjing Agricultural College|
|Keywords||Glycine max Glycine soja Hybrid Chloride ion channel gene (CLC1) Semi-quantitative RT-PCR Subcellular localization Prokaryotic expression|
The Glycine max cultivar N23674 (Lishui, Jiangsu province, the salt sensitive), the Glycine soja populations BB52 (Kenli, Shandong province, the salt tolerant) and their hybrid 4076 and 4013 (F5) strains selected for salt tolerance generation by generation were used as the experimental materials.The purpose is to analyse the role of chloride channel genes (CLCs) in improving the salt tolerance of Glycine max cultivar using salt-borne Glycine soja populations, and provide a theoretical basis for breeding the salt tolerant Glycine max cultivar with good agronomic traits and quality, which is suitable for growing under the ecological environment of coastal beach in our country.The above seedlings were stressed for 0,1,4,8,12 hours with 1/2 Hoagland solution containing 150 mmol·L-1 NaCl. The reported GmCLC1 (accession number: AY972079) cDNA sequence were used as a template, primers were designed by Primer Premier 5.0 depending on the different purposes of the experiment:semi-quantitative primers designed to compare the abundance of CLC1 mRNA of different soybean materials under salt stress through the method of semi-quantitative RT-PCR; full-length primers designed to obtain the ORF of CLC1 gene of different soybean materials, and then sequence analysis were be put up. Enzyme sites AscI and HpaI were added to both ends of the ORF of CLC1 gene to research the subcellular localization of CLC1 protein, the resulting construct of a PS1aGFP-8-CLC1 transient expression vector was introduced into onion epidermal cells via gene gun bombardment. Enzyme sites EcoRI and Xhol were added to both ends of the ORF of CLC1 gene to construct the fusion expression vector pET-30a (+)-CLC1, and then transformed it into BL21 cells and induced the foreign gene expression by IPTG. The results showed as follows:During the 12 hours of the salt-stress process, CLC1 mRNA abundance in the root of BB52 by semi-quantitative RT-PCR performed the characteristic of the continuing rapid accumulation, the response was relatively slow in the leaves, and however, it accumulated to a high level when in the 12th hours. CLC1 mRNA abundance in root of N23674 also showed the accumulation trend, but the response time delayed than BB52 (8 hours after salt treatment), the abundance was also slightly lower; it also showed a certain extent of increase in the leaves, but the performance in the salt treatment process was relatively stable.The changes in strain 4013 and 4076 were between the parent plants.The sequence alignment analysis of the experimental soybean materials showed that the cDNA full-length sequence of CLC1 gene amplified from the three soybean materials that provided by our laboratory were exactly the same, but they all had three different bases with the reported full-length sequence of cDNA of GmCLC1, and which caused the changes in two amino acids [R (Arginine) changes into L (Leucine), the former is a basic amino acid and the latter is a acidic one].The detection of reporter gene GFP after the bombardment of gene gun showed that the fusion protein under the control of the CaMV35S promoter was expressed transiently in onion epidermal cells and the GFP signal was detected exclusively in the vacuole membrane. It indicated that CLC1 is a membrane protein located in the vacuole membrane.The fusion expression vector pET30a (+)-CLCl were transformed into BL21 cells, the target gene was efficiently expressed in prokaryotic cells after the induction of IPTG.The results of semi-quantitative RT-PCR and subcellular localization indicate that, the strength of salt tolerance of different soybean materials may be closely related to chloride channel (CLC1) located at the vacuole membrane.