The Study Of Salt Sensitivity Mechanism Of Transgenic GhBASS5 Gene Arabidopsis Thaliana

Soil salinization is a worldwide agricultural problem,which seriously affects the productivity of crops.Cotton is one of the main cash crops in China,which has higher salt tolerance than other crops such as corn,wheat and rice.Nevertheless,the growth and yield of cotton were seriously affected by salt stress.Therefore,the study of the salt tolerance-related gene of cotton and breeding new varieties of salt tolerance cotton are of great significance for the full development and utilization of saline-alkali land.Na⁺ transporters in plants play an important role in regulating the accumulation of Na⁺ and responding to salt stress.Gossypium hirsutum BASS5 is a member of bile acid sodium symporter family,encoding a sodium-dependent ketone acid transporter.In the previous study,our research group found that the expression of GhBASS5 is down-regulated in response to salt stress in cotton,and silencing GhBASS5 can reduce the content of Na⁺ in cotton and improve the salt tolerance of cotton.Therefore,whether GhBASS5 has the function of promoting Na⁺ transportation to the shoots is the focus of this study.In this study,RT-PCR and qRT-PCR was performed for detection of expression level of GhBASS5 in cotton,the results showed that GhBASS5 was highly expressed in root and its transcription was down-regulated after treatment with NaCl.Instantaneous expression of GhBASS5-GFP also proved its localization in the chloroplast membrane.In order to further study the salt tolerance function of GhBASS5,we overexpressed GhBASS5 in Arabidopsis thaliana.Transgenic lines were screened by hygromycin,and transgenic GhBASS5 gene plants were detected by PCR and the expression level of GhBASS5 gene was detected by qPCR.The T4 homozygous plants of transgenic Arabidopsis were used for experiment,and wild type Arabidopsis was used as the control.The differences in salt tolerance of Arabidopsis were investigated from the aspects of seed germination under salt stress,determination of physiological and biochemical indexes at seedling stage and the accumulation of Na⁺ and K⁺.The results are as follows:1.We sowed Arabidopsis seeds on 1/2 MS culture medium without adding NaCl and added NaCl respectively,the study found that under normal condition,the germination percentage of transgenic lines and wild type Arabidopsis did not differ significantly,and under the condition of salt stress,the germination rate of transgenic lines was lower than that of wild type.2.Salt treatment of both transgenic Arabidopsis and wild type Arabidopsis showed that wild type Arabidopsis grew better than transgenic plants after salt treatment.The physiological and biochemical indexes of the leaves were determined.Under normal growth conditions,compared with wild type Arabidopsis,the content of proline,soluble sugar,MDA and O₂^-and the activity of SOD enzyme in transgenic Arabidopsis has no significant difference.The content of H₂O₂ is slightly higher than that of wild type.After salt stress,the content of proline and soluble sugar in transgenic Arabidopsis is lower than wild type,the content of MDA,O₂^-and H₂O₂ significantly higher than wild type,in addition,the SOD enzyme activity is lower than wild type,these results indicate that the wild type Arabidopsis has a salt tolerance than transgenic Arabidopsis.3.The content of Na⁺ in the leaves of transgenic Arabidopsis and wild type Arabidopsis were determined.The results showed that the content of Na⁺ in the leaves of transgenic Arabidopsis was significantly higher than that of the wild type after salt stress.In addition,the content of Na⁺ in the xylem was significantly higher than that in wild type,indicating that the overexpression of GhBASS5 increased the Na⁺ content in transgenic Arabidopsis.The above results showed that the salt tolerance of transgenic Arabidopsis was lower than wild type,and the overexpression of GhBASS5 may increased sensitivity to salt stress of transgenic Arabidopsis by promoting Na⁺ transport to shoots,damaging plastid function and the antioxidant system of plants.