Cloning And Function Analysis Of BnMYBL2 Promoter From Brassica Napus L.

Brassica napus L.is one of the most important oil crops in CHINA,with an annual planting area of about 100 million mu,providing about 55%of domestic vegetable edible oil.During the growth and development of B.napus,it is often subjected to abiotic stresses such as waterlogging,drought,low temperature,high temperature,high salinity,nutrient deficiency,and biotic stresses such as sclerotinia sclerotiorum.Stress usually decreases the growth and development of B.napus and lead to decline of quality and yield.Gene engineering is an effective way to improve plant stress tolerance.Promoters are directly related to the transcription efficiency of genes.Promoters usually are divided into constitutive promoters,inducible promoters and tissue-specific promoters.In the past few decades,the strong constitutive promoter Ca MV35S has been used to achieve good results,and a series of transgenic varieties such as storage-resistant tomatoes and insect-resistant cotton have been obtained.However,with the expansion of the application of constitutive promoters,the defects caused by constitutive promoters such as plant growth deformity and plant stunting are gradually manifested.Therefore,it is necessary to explore new promoters.The inducible promoter can not only improve plant stress resistance,but also avoid the effect of the redundant gene expression caused by the constitutive promoter on the normal growth of plants.Therefore,research and discovery of new plant-inducible promoters is of great significance.Previous work found that BnMYBL2 acted as an abiotic stress response transcription factor,which participates in a various of abiotic stress response pathways such as low temperature,drought and salt,and has strong tissue specificity.Therefore,it is speculated that BnMYBL2 promoter is a stress-induced promoter.In this study,the promoter region of BnMYBL2 gene was bioinformatics analyzed,and the promoter region of BnMYBL2 gene was isolated.The promoter expression vector was constructed and transformed into tobacco plants to analysis its response ability to stress.The main experimental results of this research are as follows:1.Bioinformatics analysis revealed that there are multiple stress response elements in the promoter region of BnMYBL2.Predicting and analyzing cis-acting elements of promoter region of BnMYBL2 with PLACE and PLANTCARE,it was found that the sequence contained anaerobic response elements ARE,low-temperature response elements LTR,light-response elements Box-4 and GT1-motif,circadian rhythm elements,hormone response element and MYB binding sites.All suggested that the promoter had the ability to respond to stress,light and hormone.2.The BnMYBL2 promoter reporter vector was constructed.It is found that BnMYBL2participates in the synthesis of plant anthocyanins and participates in the response of plants to low temperature stress.Therefore,in this study,a 834 bp promoter sequence of BnMYBL2 was cloned with PCR.p CAMBIA1305.1::5F1R::GUS plant promoter expression vector and its 5’deletion vector p CAMBIA1305.1::6F1R::GUS（deleted CAAT-box and low temperature response elements）,and transformed it into tobacco plants by leaf disc transformation method.15 T₀transgenic plants were obtained,and the T₂generation transgenic plants were identified through selfing of T₁.3.The BnMYBL2 promoter has tissue-specific and inducible expression characteristics.T₂positive transgenic plants were used as materials to analyzed the GUS transcriptional level.The results indicated that GUS gene transcription levels were significantly different among roots,stems and leaves.For 8-week-old transgenic plants,GUS gene had the highest transcription level in stem,followed by leaf,and the lowest in root;GUS gene transcription level in all tissues of 12-week-old transgenic plants was in the order of leaf>stem>root.All indicated that BnMYBL2 promoter had tissue specificity.GUS gene transcription in 4-week-old transgenic seedlings increased under the low temperature,drought,salt and ABA treatments,indicating that BnMYBL2 promoter was an inducible promoter.For 8-week-old transgenic plants,GUS transcription level decreased with the low temperature treatment for 3 h,but GUS gene transcription level increased under the treatments of drought,salt and ABA.The results showed that BnMYBL2 promoter was tissue-specific and could respond to multiple stresses simultaneously,and its transcriptional activity was closely related to plant growth stage.4.CAAT-box has an important effect on promoter activity,and the LTR element in the deleted fragment exhibits an inhibitory effect on low temperature response.Comparison of GUS transcription levels between p CAMBIA1305.1::5F1R::GUS transgenic plants and p CAMBIA1305.1::6F1R::GUS transgenic plants,it was found that under non-stress conditions,the GUS gene transcription levels in p CAMBIA1305.1::5F1R::GUS plants is significantly higher than that of p CAMBIA1305.1::6F1R::GUS transgenic plants,indicating that the presence of CAAT-box in the deleted fragment has a great influence on the transcriptional activation efficiency of the promoter.And it may cause the activation or suppression function of the promoter to be amplified in response to low temperature stress.Under low temperature stress,the deleted fragment responds to induce activation of GUS gene transcription,while the non-deleted fragment shows inhibition.