| Salt injury is a main cause for crop yield reduction. To cultivate salt-tolerant transgenic crops is a rapid and effective method to resolve this problem. But effective genes scarcity is the obstacle to the development of gene engineering on salt-tolerance. The main aim of this research is just to find out new wheat salt-tolerant genes and to study their functions. It is hoped to offer some useful reference to elucidate the salt-tolerant mechanism of plants and obtain salt-tolerant transgenic wheat with high yield.In our research,we use salt-resistant mutant RH8706-49 and salt-sensitive mutant H8706-34 in wheat (both lines were single-seed descent lines obtained by hybridization of Punong 3665/ Bainong 3039 F1, anther culture, EMS mutation, repeated salt stress selection, and stably cultured for 15 generations. The salt-resistance index of RH8706-49 and H8706-34 are respectively no less than 1.3 and no more than 0.5) In this paper,we analyzed the expression pattern of wheat Vacuolar proton-ATPase genes (A,B,C,D,F,G,H,a,c,d) on 1% NaCl and 50μMABA stress, which have some differences in salt tolerance between salt-resistant mutant RH8706-49 and salt-sensitive mutant H8706-34.We also cloned V-H~+-ATPase B,F,G,H and c subunits and further transfer wheat V-H~+-ATPase B subunit into Arabidopsis thaliana to study salt tolerance of transgenetic plants. The experiments were shown as follows:1 cloning and sequence analyzing of subunits B,F,G,H and c of vacuolar H~+-ATPase in wheatwheat vacuolar H~+-ATPase B,F,G,H and c subunits logic sequences were obtained according to the other plants published vacuolar H~+-ATPase subunits B,F,G, H,c sequences (rice,barley etc.) by Blast in NCBI. All of these full cDNA sequences including vacuolar H~+-ATPase subunits B, F, G, H and c were cloned respectively.The cDNA of subunit B was 1467bp long, coding 488 amino acids with molecular weight of approximately 54.0 kDa which is a relatively conservative subunit. Phylogenetic analysis showed that the V-H~+-ATPaseB of wheat had high homology with other reported V-H~+-ATPase subunits, such as barley and rice. The cDNA of subunit F was 393bp long, coding 130 amino acids with molecular weight of approximately 14.4 kDa. The cDNA of subunit G was 333bp long, coding 110 amino acids with molecular weight of approximately 12.4 kDa. The cDNA of subunit H was 1359bp nucleotides long, coding 452 amino acids with molecular weight of approximately 51.4 kDa. The cDNA of subunit c was 498bp long, coding 165 amino acids with molecular weight of approximately 16.6 kDa. Amino acid sequences alignment analysis suggested that the V-H~+-ATPase B,F,G,H and c subunits of wheat had high homology with barley and rice and have 95% similarityAfter we got these gene's sequences, we can detect V-H~+-ATPase subunits in wheat how to function completely and deeply.2. Expression of wheat V-H~+-ATPase subunits under different stress conditionsTo understand the relation of V-H~+-ATPase and abiological stress, the expression profile of these V-H~+-ATPase subunits in root and leaves under stress including 1% NaCL and 50μM ABA(0,1,6,12,72 h) were analyzed for both salt-resistant and salt-sensitive wheat lines, by Real-Time PCR.2.1%NaCLfor the salt-resistant mutant 49, after salt stress, the expression levels of root V-H~+-ATPase subunits were evidently higher than those before stress, with an overall trend of increase as stress time prolonged.In the root of salt-sensitive mutant 34, the post salt stress expression levels of V-H~+-ATPase subunits were significantly lower than their pre-stress levels. But in leaves of mutant 34,the overall post salt stress expression levels of all the subunits of V-H~+-ATPase were slightly higher than their pre-stress levels.2.2 50μM ABAIn the root of salt-resistant mutant 49,the overall expression levels of V-H~+-ATPase subunits after 50μM ABA stress were slightly higher than pre-stress levels at 6 h and 12 h but were lower than pre-stress levels at other time points. The overall post-stress expression levels of the subunits were higher than pre-stress levels,and peaked at 24 h,which was consistent with the trend under salt stress. In the root of salt-sensitive mutant 34, the overall post ABA stress expressions of V-H~+-ATPase subunits in root were lower than their pre-stress levels, and n leaves of mutant 34, the post stress expression levels of the subunits were generally higher than their pre-stress levels and had two expression peaks at respectively 1 h and 24 h; the overall trend was consistent with that in salt stress.When salt-resistant mutant 49 were treated with abiological stress, the expression levels of these subunits (A, B, C, D, F, G, H, a, c, d) were higher. With increasing the expression of V-H~+-ATPase genes, the salt tolerance capability of plants was enhanced.3. Salt tolerance research of Wheat V-H~+-ATPase BTo confirm the function of Wheat V-H~+-ATPase B gene, we constructed the binary vector and transformed Arabidopsis thaliana by using floraling dip method. After treat with salt stress, the transgenic plant's germination rat ,growth and survival status are better than the Control plants in Medium that contains NaCl; the transgenic plant not only can improve normal growth of root but also increased salt tolerance of plant. We deduce Wheat V-H~+-ATPase B gene play an important role in plant salt tolerance. |
PDF Full Text Request