| Raspberry is a small berry fruit tree with extremely high nutritional value.Abiotic stresses,such as drought,high salt and low temperature can affect the growth,yield and quality of raspberry fruit.As one of the five major hormones in plant,abscisic acid(ABA)plays important role s in response to plant abiotic stress and regulation of fruit ripening.9-cis-epoxycarotenoid dioxygenase(NCED)is a key rate-limiting enzyme in ABA biosynthesis pathway.The raspberry fruits of ‘Heritage' were used as the materials in this study and the RiNCED1 and RiNCED2 genes were obtained by PCR amplification from ‘Heritage'.The plant super-expressing vectors of RiNCED1 and RiNCED2 were constructed and transformed into Arabidopsis by Agrobacterium tumefaciens-mediated method to obtain positive super-expressed Arabidopsis plants.The Arabidopsis plants were treated with high salt,low temperature and drought stress and the physiological indicators were measured to analyze the role of RiNCED1 and RiNCED2 in the abiotic stresses of raspberry.The aim is to provide genetic resources for the cultivation of fruit trees resistance to abiotic stresses in Rubus or other fruit trees in the future,and also lay a foundation for later work to verify their function of NCED in regulating fruit ripening.The main results of the work are as follows:(1)The open reading frame of RiNCED1 gene was 1848 bp,encoded a total of 615 amino acids,MW = 68.039 k Da,p I = 7.26,and the total average hydrophilic coefficient is-0.369.The open reading frame of the RiNCED2 gene was 1821 bp in length and encoded 606 amino acids in total.MW=66.478 k Da,p I =6.24,total average hydrophilic coefficient is-0.291.The multi-sequence alignment analysis by DNAMAN showed that RiNCED1 and RiNCED2 encoded proteins were highly similar to the NCED proteins of other species.The conserved domains of RiNCED1 and RiNCED2 protein were analyzed.There was a conserved RPE65 domain which belonged to NCED family in each of the proteins.(2)The leaves of wild-type Arabidopsis and Arabidopsis transformed with empty vector were yellowed and wilted seriously after treated with 200 m M NaCl solution for high salt stress.But leaves of most transgenic Arabidopsis plants were still green.When returning to normal conditions,the survival rate of Arabidopsis plants transformed with RiNCED1 or RiNCED2 gene was significantly higher than wild-type Arabidopsis and Arabidopsis transformed with empty vector.After low temperature treatment at-4 ? for 10 h,wild-type Arabidopsis thaliana and Arabidopsis thaliana transformed with empty vector suffered severe freezing damage than transgenic Arabidopsis thaliana.When the Arabidopsis thaliana plants were removed to normal conditions,most of the Arabidopsis plants transformed with RiNCED1 or RiNCED2 gene were alive.Under drought condition,wild-type Arabidopsis thaliana and Arabidopsis transformed with empty vector showed significantly lower survival rate and more leaves were wilted than transgenic Arabidopsis plants.(3)The ABA content of Arabidopsis plants was determined by high-performance liquid chromatography.The results showed that the ABA content of transgenic Arabidopsis was significantly higher than that of wild-type Arabidopsis and Arabidopsis transformed with empty vector no matter under normal or stress conditions.The indicated that overexpression of RiNCED1 or RiNCED2 could increase the content of ABA in plants,thus demonstrating that these genes were involved in ABA biosynthesis.(4)Under abiotic stresses,except for malondialdehyde(MDA)content,the other physiological indicators such as chlorophyll content,proline content,activities of superoxide dismutase(SOD),peroxidase(POD)and catalase(CAT)in Arabidopsis thaliana plants transformed with RiNCED1 or RiNCED2 was significantly higher than that of wild-type Arabidopsis and Arabidopsis transformed with empty vector.This indicated that transformation of RiNCED1 or RiNCED2 gene increased the tolerance of Arabidopsis plants to high salt stress,low temperature stress and drought stress. |
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