Cloning And Functional Analysis Of Tobacco PRE1

Tobacco belongs to Nicotiana of the Solanaceae family,which has extremely high medicinal and economic value.The tobacco industry has also made great contributions to China's fiscal revenue.The yield and quality of tobacco directly affects its value.When tobacco changes from vegetative growth to reproductive growth,most of nutriment will be transported to the top,and the growth of leaves,stems and roots will slow.The main value of tobacco depends on the leaves.In order to make tobacco to keep vegetative growth,topping is used to remove the apical dominance of tobacco,block the transfer of nutriment to the reproductive organs,increase the distribution of nutrients in the leaves,promote the accumulation of dry matter in tobacco leaves,and increase the yield and quality of tobacco leaf.Tobacco plants have many responses to topping,including the changes of phytohormone source,the second roots growth,and the increase of nicotine content.The tobacco bHLH(basic/helix-loop-helix,bHLH)transcription factor PRE1(paclobutrazol resistance 1)can integrate a variety of hormone signals to regulate cell elongation in plants.We had found that the expression level of PRE1 was significantly increased after tobacco topping,suggesting that it is involved the growth of leaves and the elongation of roots.In order to explore its role in tobacco growth and development,NtPREl was cloned from the variety K326(Nicotiana tabacum L.cv K326),and the main results are as follows:(1)Through bioinformatics analysis,it was found that NtPREl belongs to PRE subfamily of the tobacco bHLH family.NtPRE1 has a full length of 1931 bp and encodes a 93 amino acid polypeptide.NtPREl has a typical helix-loop-helix tertiary structure of bHLH,but has no DNA binding domain.It can bind to other proteins to indirectly regulate plant growth and development.There are many cis-acting elements related to abiotic stress in promoter of NtPRE1,including GA,MeJA,IAA,ABA,drought and low temperature responsive elements,which indicates that NtPRE1 can integrate multiple hormone signals to regulate plant growth and development.(2)NtPREl is tissue-specifically expressed in different growth stages of tobacco.When tobacco was treated with GA,IAA and topping,the expression level of PRE1 was up-regulated.While the expression level of PRE1 was down-regulated under MeJA,ABA,mechanical and drought treatment.The results showed that NtPREl may participate in different signaling pathways and play an important role in plant growth and development.(3)To study the function of NtPRE1,the overexpression and silencing vectors of PRE1 were constructed,transformed to tobacco K326 with Agrobacterium mediated transformation method.After the transgenic plants were verified at DNA and RNA levels,their phenotypes were analyzed.The roots growth of transgenic plants was faster than non-transgenic plants,and the narrow and long leaves present in transgenic plants.The relative expression of genes related to plant cell elongation,such as XTH31,EXPA1,EXPA2,RLK,and LTP,was also increased accordingly in transgenic plants,indicating that NtPRE1 activates the expression of multiple genes to promote tobacco growth and development after the integration of multiple hormone signals.