Cloning And Functional Identification Of Cassava MeSCL30

Cassava?Manihot esculenta Crantz,cassava?is one of seven crops in the world and is the main staple food crop in the tropic area.However,the yield of cassava is seriously affected by drought,salinity,low temperature and other environmental factors.Previous studies have shown that alternative splicing is involved in many physiological processes and is an important post-transcriptional mechanism when encountered with abiotic stress.Serine/arginine rich proteins?SR?are important splicing factors,which play an important role in response to abiotic stress in planta.Bioinformatics analysis showed that Me SCL30 is one of members in cassava SR protein family.Therefore,the purpose of this study is to analyze the important function of Me SCL30.The main results are as follows:1. Coding regions of eighteen cassava SR protein were cloned and plant expression vectors were constructed by gateway system.The expression vectors were transformed into Arabidopsis thaliana by the Agrobacterium tumefaciens-mediated floral-dip method to screen homozygous lines for subsequent phenotype analysis.2. Bioinformatics analysis showed that full length of Me SCL30 gene was 816 bp and encoded 271 amino acids.The Me SCL30 protein was rich in 53 positively charged Arg residues.The Me SCL30 protein was about 31 KD and the isoelectric point was 11.16.Me SCL30 was a hydrophilic basic protein and contained 7 exons and 6 introns.Me SCL30protein contains RRM domain and RS domain,and had the closest relationship with At SCL30.We then analyzed the cis acting elements in the promoter region of Me SCL30gene,and found that many elements related to abiotic stress,for example,heat stress response element,low temperature response element and drought response element,this result indicated that cassava Me SCL30 may participate in the response of stress.3. The results of subcellular localization showed that GFP was not expressed and therewas no fluorescence signal in the cells when p GWB505?C-GFP?was transferred into tobacco.When p GWB505:Me SCL30 was transferred into tobacco,the fluorescence signal could be detected in the nucleus,indicating that Me SCL30 was a nuclear localization protein.Under the conditions of sorbitol,the fluorescence signal decreased,indicating that the expression of Me SCL30 protein was down-regulated,and the stress inhibited the translation process or promoted the degradation process.4. The results of real-time fluorescent quantitative PCR?q RT-PCR?showed thatMe SCL30 was expressed in roots,stems and leaves of cassava,and the highest expression level in stems was found.Under the conditions of sorbitol,dehydration,ABA and Na Cl,the expression level of Me SCL30 was significantly up-regulated or down-regulated,indicating that Me SCL30 may be involved in the regulation of cassava response to abiotic stress.5. Two Arabidopsis transgenic lines with the highest expression were selected by q RT-PCR,OX8 and OX11.The results showed that the germination rate,greening rate,root length and fresh weight of transgenic Arabidopsis were significantly higher than those of wild type under sorbitol stress.Under ABA stress,the germination rate,greening rate,root length and fresh weight of transgenic Arabidopsis were significantly lower than those of wild type.These results showed that over-expression Me SCL30 Arabidopsis had stronger drought resistance than wild type Arabidopsis.6. We counted the survival rate of over-expression Arabidopsis and wild typeArabidopsis after drought treatment,and measured the content of chlorophyll and anthocyanin.The results showed that the survival rate of over-expression Me SCL30Arabidopsis was significantly higher than wild type Arabidopsis after drought treatment.The content of chlorophyll in the leaves of over-expression Me SCL30 Arabidopsis was much higher than wild type Arabidopsis,however,the content of anthocyanin in wild type Arabidopsis was higher than over-expression Me SCL30 Arabidopsis.7. The results of enzyme activity test showed that the catalase?CAT?activity andperoxidase?POD?activity of over-expression Me SCL30 Arabidopsis were higher than wild type Arabidopsis,and the content of hydrogen peroxide?H₂O₂?was lower than wild type Arabidopsis.This result indicated that over-expression Me SCL30 could reduce oxidative damage and improve drought resistance.8. The response process of Me SCL30 gene to abiotic stress and ABA on the level ofalternative splicing was studied by reverse transcription polymerase chain reaction?RT-PCR?.The results showed that abiotic stress and ABA had different effects on the splicing process of Me SCL30 gene.9. We used 300 m M sorbitol to treat over-expression Arabidopsis and wild type Arabidopsis at a similar developmental stage.The results of q RT-PCR showed that the expression levels of drought stress response related genes NCED3 and RD29A in the two over-expression lines were significantly higher than the wild type Arabidopsis.