SsCMO Gene Isolation From Suaeda Salsa L.and Genetic Transformation Into Soybean

Soybean (Glycine max L.Merr) is an important economic crop in international trade due to its high protein and fat contents. Soybean growth and yield are susceptible to external en-vironmental stresses, such as salinity, drought, frost and so on. Breeding of stress-tolerant soybean lines in a direct and effective way, i.e. gene transformation will make a great contribute to solving the crisis in soybean industry in our country.In this paper, firstly we cloned a stress-tolerant related gene and introduced it into different soybean varieties. Specifically, seeds of Suaeda salsa L., a halophyte growing on the tidal flat in Zhejiang Province were sampled and then planted in the green house of the Institute of Crop and Nuclear Technology Utilization. Seedlings were subject to6%NaCl treatment for RNA extraction and further gene cloning. A1332bp coding sequence for CMO gene was amplified from the cDNA of Suaeda salsa L. using homologous cloning technology. A564bp5’flanking sequence and a364bp3’flanking sequence were also amplified by TAIL PCR. This SsCMO gene was further cloned into plant express vector pBA002using conventional methods. The recombination plasmids were then introduced into Agrobacterium EHA105by freezing-melting transformation method. Subsequently, SsCMO gene was introduced into the soybean varieties’zhechun NO.3’and’nannong88-31’ via Agrobacterium mediated embryonic tip transformation method. As the second part of the paper, effects of subjecting exogenous glycine betaine on soybean seeding under NaCl stress were also studied. The main results of this paper were as follows:SsCMO gene was cloned from Suaeda salsa L., with the full length cDNA of2260bp. The ORF of SsCMO is1332bp in length, encoding443putative amino acid residues. This CMO protein composited of20kinds amino acid and had isoelectric points of6.64.Compa- red SsCMO with other CMO amino acid sequence, found those sequence in162-167and290-296areas there are two conservative regions. Clustering analysis indicated that SsCMO is highly homologous with Liaoning SICMO gene.The SsCMO gene was further cloned into plant express vector pBA002using conventional methods. The recombination plasmids were then introduced into Agrobacterium EHA105by freezing-melting transformation method. Subsequently, SsCMO gene was introduced into the soybean varieties’zhechun NO.3’and’nannong88-31’via Agrobacterium-mediated embryonic tip transformation method. After preliminary screening with10mg/L Basta coating on seedling leaves, the basta-resistant regenerated plants were further confirmed by polymerase chain reaction (PCR) analisis. The average transformation frequence in this study were1.2%.Effects of exogenous glycine betaine on some physiological indexes of soybean variety Zhechun NO.3seedlings under NaCl stress were studied. The photosynthetic pigments, proline, malondialdehyde accumulation (MDA), soluble sugar, relative electric conductivity and root activity of soybean seedlings were investigated. The results showed that exogenous betaine significantly increased the photosynthetic pigments and soluble sugar content, decreased proline and MDA accumulation, reduced the relative electric conductivity of soybean leaves, and enhanced root activity of soybean seedlings, which indicated that the application of exogenous betaine could enhance the photosynthetic activity of soybean leaves, improve the osmoregulatory ability of seedlings, remit the damages caused by NaCl stress, and then improve the salt tolerance ability of soybeans.