Cloning And Expression Analysis Of HbNAC1,HbNAC2, HbNAC3, Three Hevea Brasiliensis Transcription Factors

NAC (NAM andATAF1/2, and CUC2) is found as a class of plant-specific transcription factor family for more than10years, involving in plant growth and development, aging, hormonal response, secondary metabolism and responses to stress. A highly conservative NAC domain in N-Terminal is the main structural characteristics of all NAC transcription factors containing about150amino acid residues. Natural rubber is a secondary metabolite of the rubber-producing plants.Hevea brasiliensis is the main source of natural rubber. In order to investigate the molecular mechanism of ethrel-stimulated latex yield in H. brasiliensis, we constructed an ethrel-induced latex SSH cDNA library from latex, in which three ESTs, HbEST30, HbEST31and HbEST32with high homology to NAC transcription factor genes were identified. Here, three NAC genes were cloned and characterized in latex and their expression was examined in different organs and under ethrel stimulation. The results were as follows:1.According to the EST sequences, threes cDNAs of HbEST30-, HbEST31-and HbEST32-corresponding genes were cloned in the latex fromHevea brasiliensis by RACE and RT-PCR techniques.Bioinformatics analysis showed that the related HbEST30gene cDNA full length1419bp and contains a full open reading frame, encoded with309amino acid residue peptide, the HbEST31-corresponding gene cDNA is1257bp with a762bp open reading frame and encodes the254amino acid residue peptide, and the HbEST32-corresponding gene cDNA is1131bp with a876bp open reading frame and encodes the289amino acid residue peptide. Structural analysis indicated that the typical NAC transcription factor-specific domain is in the deduced proteins corresponding to three genes, desiganated as HbNACl, HbNAC2and HbNAC3respectively.2. Multiple alignment showed that the amino acid homology is respectively17%between HbNAC1and HbNAC2,11%between HbNAC1and HbNAC3, ans34%between HbNAC2and HbNAC3amino acid homology is17%and34%. The NAC typical domain exits in all tress proteins, but the location and number of amino acids in NAC domain varies in HbNAC1, HbNAC2and HbNAC3. HbNAC1shares79%.80%.57%and60%identy with NAC from Populus trichocarpa, Ricinus communis, Oryza sativa and Arabidopsis thaliana respectively. HbNAC2shares90%,87%,81%,77%and74%identy with NAC from Ricinus communis,Populus trichocarpa, Passiflora morifolia,Citrus trifoliataand Malus x domestica respectively.HbNAC3shares89%,85%,75%,77%and75%identy with NAC from Ricinus communis. Populus trichocarpa,Glycine max,Medicago truncatula and Arabidopsis thaliana respectively. 3.We used the genomic of leaves as template, cloned three genes respectively. The results showed that, the genomic sequence and cDNA of HbNAC1were the same, it indicated that HbNAC1contained no intron; the length of HbNAC2genome sequence was1554bp, it contained three exons and two introns; the length of HbNAC3genome sequence was1725bp, and also contained three exons and two introns. The genome structure of HbNAC2and HbNAC3was the typical structure of NAC family, that was the structure of three exons and two introns.4.We constructed phylogenetic tree of21NAC proteins from different plants. The results showed that, the evolutionary distance between HbNAC2/HbNAC3and ATAF were near. ATAF responded to stress such as drought stress, therefore we speculated that HbNAC2/HbNAC3may also play a role in stress such as drought response; the evolutionary distance between NAC proteins HbNAC1and RCOM₁172140were the nearest, but whether it involved in the regulation of the biosynthesis of natural rubber latex was unknown.5.The5'regulatory sequence of HbNAC1and HbNAC3, the length of which were514bp and1363bp respectively, were cloned from the genomic DNA of H. brasiliensis leaves by GenomeWalking method. Besides the TATA-box and CAAT-box, there were several promoter elements and many cis-regulatory elements related to abiotic stress response such as stem tip specific positive cis-regulatory element, CGTCA-motif responded to JA, light response element ACE, high temperature stress response element, TC-rich repeats, ethylene response element located on5'regulatory sequence of HbNAC3etc. Among these elements, the last one was more interesting.6.The expression characteristics of HbNAC1/2/3gene were investigated by semi-quantitative RT-PCR analysis. The results demonstrated that the expression profiles of HbNAC1/2/3of H. brasiliensis were constitutive expression, but these genes expressive quantity in different tissues and organs was significantly different. The expression of HbNAC1was strongest in latex, following was in leaf and flower, which in bark and root was weak. The expressions of HbNAC2t in flower, root and bark were the same, which in latex and leaf were stronger. The expressions of HbNAC3in bark and latex was weaker than that in leaf, root and flower. The ethephon stimulation effect on the expression of three genes in latex was detected. The result shows that, there was no significant change in expression quantity of HbNAC1after3h ethephon treatment, but sharply increased after12h treatment, and still maintain in high level after24h. After3h ethephon treatment, the expression quantity of HbNAC2decreased significantly, while after24h treatment, it could not reach to the expression quantity level of untreated. On the contrary to HbNAC2, the expression quantity of HbNAC3start to increase after3h treatment, and still maintain in high level after24h treatment.7. pET30a-HbNAC1/2/3were constructed successfully. Corresponding protein was obtained through prokaryotic expression and protein purification which laid the foundation for further screening target gene. Over-expression of plant expression vector of pCAMBIA1304-HbNAC1was constructed successfully. Positive transgenic tobacco obtained by Agrobacterium tumefaciens-mediated leaf disc transformation method. After PCR detection, the genome of7tobaccos contained HbNACl gene, which laid the foundation for in-depth study of the physiological functions of HbNACl gene.Conclusion: This study has successfully isolated and identified three genes of NAC transcription factors, named HbNAC1,HbNAC2and HbNAC3. The expression of HbNAC1/2/3were constitutive expression and tissue-specific. HbNAC1and HbNAC2expressed strongly in latex, while HbNAC3expressed strongly in flower, leaf and root. ET could regulate the expression of HbN AC1/2/3in latex. HbN AC1/2/3were a kind of multi-functional transcription factor in H. brasiliensis. The expression quantity of HbNACl and HbNAC3increased significantly after ET treatment, while HbNAC2decreased, this suggested that HbNAC1/2/3might take part in ethylene signal transduction.