Analysis Of Stress-resistance Function Of 14-3-3 Genes HaFT-1 And HaFT-9 In Haloxylon Ammodendron

Haloxylon ammodendron is a Haloxylon Chenopodiaceae,a kind of Perennial shrub or small tree.Long-term survival in gravel desert,clay desert,fixed and semi-fixed sandy land and saline-alkali land has strong ecological adaptation characteristics,such as drought resistance,cold resistance,salt-alkali resistance,high temperature resistance,etc.To study the molecular mechanism of stress tolerance of Haloxylon ammodendron is of great significance for breeding and screening resistant Haloxylon ammodendron plants and accelerating the process of desertification control and ecological control in China.Our research group previously cloned and discovered nine genes of Haloxylon ammodendron 14-3-3 protein.The genes were named HaFT-1 to HaFT-9 according to the initials in the Latin name of Haloxylon ammodendron and Fourteen and Three in the 14-3-3 protein name,respectively.HaFT-1 and HaFT-9 were identified as the research objects by comparative analysis.The understanding of the mechanism of H.ammodendron stress resistance was deepened by yeast two-hybrid,subcellular localization and functional validation of transgenic Arabidopsis.thaliana The results were as follows:1.In this experiment,the yeast two-hybrid vector was constructed by Gateway technology,and the possible interaction between HaFT-1 and HaFT-9 was studied.The results showed that there was no interaction between HaFT-1 and HaFT-9 protein monomers.HaFT-1 did not interact with itself.HaFT-9 had strong interaction with itself.2.Transient expression vectors of 35S::HaFT-1-GFP and 35S::HaFT-9-GFP were constructed using pCAMBIA1304 as vector.Transforming onion inner epidermis cells to observe green fluorescent protein signal.The results showed that the proteins of HaFT-1 and HaFT-9 were located in the nucleus,which was consistent with the predictions of BaCelLo online software.3.The plant expression vectors of HaFT-1 and HaFT-9 were constructed using pCAMBIA3301 as vector.The target gene was transferred into Arabidopsis thaliana by inflorescence infection.The DNA of T1 transgenic plants was extracted and verified by PCR.T2 lines were identified by 3:1 segregation ratio using resistant plate,screened from multiple generations to T3 generation and homozygous lines were selected using resistant plate.The results showed that eight transgenic lines were obtained from each of HaFT-1 and HaFT-9 by resistance screening and PCR validation,and a total of three of these lines did not meet the 3:1 segregation ratio.Transgenic homozygous plants with high expression of HaFT-1 and HaFT-9 in T3 generation were selected by resistance plates for determination of germination rate and physiological parameters under high salt and drought stress in Arabidopsis thaliana.The germination rate of salt-and drought-stressed transgenic Arabidopsis thaliana seeds was lower than that of wild-type Arabidopsis thaliana,and the sensitivity of HaFT-1 transgenic Arabidopsis thaliana under stress was higher than that of HaFT-9 transgenic Arabidopsis thaliana.High-salt and natural drought stress were applied to 30 day-old Arabidopsis thaliana.Under 250 mmol/L salt stress,the salt tolerance of transgenic HaFT-1 plants was significantly lower than that of wild-type Arabidopsis thaliana,while the salt tolerance of transgenic HaFT-9 was not different from that of wild-type Arabidopsis thaliana.