Fine-stem Stylo SgiMYB Gene Cloning And Cold Resistance Analysis Of Transgenic Plants

Stylosanthes guianensis was a kind of important leguminous forages,which has high stem and leaf yield,rich in nutritional value,rich in amino acids and vitamins.At the same time,it is widely used in animal forage,hay meal production,and it can also be used to soil nitrogen fixation,restore the desert.However,the temperature below 10 ? can inhibit the normal growth.In the process of breeding of thin-stem stylo(S.guianensis var.intermedia),a new strain "89-1" was obtained.Using a new generation of Illumina sequencing technology,the transcriptome sequencing was conducted in the new strain at low(4?)and warm(28?)temperature.The basis of the transcriptome sequencing,the SgiMYB gene was cloned and analysised function.The results were as follows.(1)According to the transcriptome sequencing results,RNA and cDN A of SgiMYB gene was extracted and synthesised.The full length SgiMYB c DNA is 879 bp.The N terminal of SgiMYB protein has a conservative SANT domain structure,which belongs to the SANT subtribe.SgiMYB amino acids sequence was aligned and analysised,indicating that SgiMYB gene belonged to CCA1 group in MYB-related type MYB transcription factors family.(2)It was proved that SgiMYB gene had a strong transcription activity,and located in the cell nucleus by building a single yeast hybrid vector(p GBKT7-SgiMYB)and subcellular localization vector(pAN580-SgiMYB).The qPCR analysis results of SgiMYB gene identified that SgiMYB expression quantity rapidly rised under the 2 h treatment of 4?,but not significantly changed with treatment time prolongation.The gene expression quantity of different tissues was leaf > stem > root.(3)Transgenic tobacco plants(O E-3,and OE-4 OE-5)were obtained using the method of agrobacterium mediated transformation with pBA002–SgiMYB vector.Under the condition of low temperature treatment of 24 h and 48 h,transgenic plants had higher cold tolerence than the wild plants.And SgiMYB gene expression of transgenic plant was significantly higher than that of wild type.The various of physiological indexes were detected in the transgenic tobacco s,and results showed that MDA and relative electrolytic leakage were significantly higher than wild type tobacco after 12 h in cold condition.The content of proline and soluble sugar was higher than the wild type after 6 h in low temperature treatment,but appeared significant difference after 24 h of cold treatment.These results indicated that the overexpression of SgiMYB gene can improve the cold resistance of plants.Under the low temperature treatment,the quantitative analysis of downstream cold resistance genes(C BF1,CBF3,ERD10)of transgenic plant OE-3 and wild type was conducted.As a result,the CBF1 gene demonstrated a significantly higher expression than the wild type in cold treatment of 2 h,12 h and 24 h.The CBF3 gene expression quantity of O E-3 was less than that of wild type in low temperature treatment of 6 h,and was obviously higher than wild type after 12 h and 24 h cold treatment.However,ERD10 gene expression of OE-3 was significantly higher than that of wild type in different cold treatment times.These results suggested that SgiMYB gene was involved in cold resistance regulation,and it had direct or indirect regulation to cold resistance gene expression,which improve the cold resistance ability in plants.