Transformation Of GsCHX19 Gene Into Medicago Sativa L. And Cultivation New Strains

There are 99.13 million of hectares saline-alkali land ar ea in our country, and gr adually expand the tr end in recent years. The r esource of ss ilage is ser ious shortage along with the rapid development of animal husbandr y that lead the demand of feed and food grain s ignif icantly r is ing. Alfalf a(Medicago sativa l.), known as the "king of the grass" reputation, it is important as forage crops, also important crop in impr oving soil. With the use of genetic engineer ing technology and the development of increas ingly mature, the study of transgenos is alfalf a also has a good momentum of development.It is used by the method of agr obacter ium mediated alfalf a leaf festival with its easy oper ation, high eff ic iency, low copy number, exogenous gene integr ation to overcome the shortcomings of tra ditional genetic br eeding.T his method is one of the most effective means to cultivate res istanc e new strain. It can make full use of saline- alkali land resources in China and r aise the yield of alfalf a at the s ame time.It has gr eat s ignif ic ance to the development of animal husbandry and improvement of ecological environment.CHX(cation/H+exchanger) is the plant body exists in cytoplasm membrane or vacuole membr ane,such as coding Na+, K+/ H+ revers e trans porters of gene families.CHX gene families belong to CPA(cation/proton antiporter) superfamily. CPA super family by Na+, Li+ or K+ H+ to regulate cellular cation exchange and p H steady state.Prokar yotes and eukaryotes CPA super family mainly inc lude two categor ies, CPA1 and CPA2. CPA1 which in the plant body and Nha P which in bacter ia have high sequence s imilar ity, inc luding more widely research of NHX(Na+/ H+ exchanger) family. Relatively less res earch of CPA2 family inc luding KEA( K+ efflux antipor ter) and CHX,CHX w ith KHA1, Nha S4 of Synechocystis in yeast has a high sequenc e similar ity.The present study showed that the CHX pr otein only exis ts in advanced plants, about 800 amino ac ids, including about 400 res idues at Na+/ H+ exchanger of the end of the N hydrophobic and 300~400 hydrophilic str uctural domain of the end of C terminal.Due to the hydrophilic carboxyl term inal does not contain known conservative structur e domain, so its function is unknown. But not all the CHX have such featur es.Predict Nha S4 pr otein from the bacter ia, for example, encoding 715 amino acids, in fact only code 410.Our laboratory has been with independent intellectual property r ights in the initial stage and play an impor tant role in the abiotic stress respons e of GsCHX19 gene and synthetic p CAMBI A330035 S u construction of plant express ion vector, with the method of agrobacter ium mediated transformation of alfalf a, obtained the transgenic plants.To deter mine the new comprehens ive impr ovement of salt-toler ant alf alf a new strains through bioinformatics meth od and molecular biology method. This study not only in order to establish the system of molecular breeding technology of alfalf a, pr ovides an important res earch value, and f or the f ull development and utilization of saline- alkali land r esources in our country, improve the yield and quality of alfalfa, opened the broad development prospect. The main results were as follows:(1)Bioinfor matics analysis of GsCHX19 geneGsCHX19 gene is alkali str ess response genes that the lab has obtained in pr evious studies, the CHX19 gene der ived from w ild soybean alkali stress transcriptome data, the gene res pond t o alkali stress actively in transcr iption.We speculated that it may be actively involved in the str ess signal tr ansduction pathway, has impor tant function in the alkali str ess path. By the method of bioinf ormatics, analys is the gene encoding pr otein for the phys ical and chemical pr operties, feeling of water-based, phosphor ylation sites, across a membrane structure domain and functional annotation of bioinf ormatics analys is, domain, and predict its protein structur e pr ediction the gene upstream pr omoters r e lated regulation of osmotic str ess components, and provide the bas is for further research.(2)Transformation GsCHX19 gene into alfalfa and acquisition the resistance plantZhaodong alf alf a as receptors mater ials, us ing the method of agrobacter ium mediated. GsCHX19 gene( have been with independent intellectual proper ty r ights, play an impor tant r ole in the abiotic stress respons e)and synthetic p CAMBI A330035 S u construct plant express i on vector to genetic transfor mation of alfalf a, then transf ormat the recepto r for alfalfa lea f section, 20 strains of resistant plants were obtained through the regeneration system.(3)Molecular biology detection the plant that transformed with GsCHX19 gene20 strains GsCHX19 gene res istance plants conduct P CR detectio n and get PCR pos itive plants 16 strains, PCR pos itive rate is 80%; Rt-pcr detection in the PCR pos itive plants, received 11 str ains GsCHX19 overexpr ession at the tr anscriptional level of plant genes, RT-pcr pos itive rate was 68.75%.