| Abiotic stresses severely affect quantity and quality of crop product. Chickpea is the third most important legume crop in terms of cultivation area, and is grown primarily in the arid and semi-arid regions of the world. Through long-term evolution and adaptation to extreme conditions, chickpea has been found to be rich in resistance genes for arrange of abiotic stresses, including drought and cold, and has been suggested as amodel plant for investigation of physiological mechanisms of plant development and responses to stresses. We have cloned 6 NAC genes from the drought-related cDNA library constructed using the stress resistance germplasm from Xinjing, which named CarNAC1～6. All the six NAC genes harboured typical NAC domain, and could be significantly induced by various stress and hormone signals. In recent years, many NAC members have been found playing important roles in plant growth, development and stress responses, are taken as good genetic resource for the improvement of crop tolerance to environmental stresses. We selected CarNAC4, Car NAC5 and Car NAC 2 for further research, which containing analysis of expression patterns under induction of stresses, analysis of promoter sequences and location of c/s-acting elements, analysis of binding ability with DNA fragments. Function of three NAC genes were analyzed using transgenic Arabidopsis. Main results of this study are as follows:1. Expression patterns of CarNAC4, CarNAC5 and CarNAC2 genes were investigated under diverse stress and chemical treatments. CarNAC4 gene not only was significantly induced by drought, high salinity, high temperature and low temperature, but also was significantly up-regulated by abscisic acid (ABA), methyl jasmonate (MeJA), indole-3-acetic acid (IAA), and H2O2. Expression of CarNAC5 gene was strongly induced by drought and high temperature treatments and IAA treatment. Expression of CarNAC2 gene was only induced by drought and ABA treatments. These findings suggested the three CarNAC genes may act synergistically in response to stress and hormone signals, although they showed different patterns.2. By Genome Walking technology, promoter sequence of of CarNAC4 and CarNAC5 were isolated and submitted into promoter datebase for cis-acting elements predication. The results showed that many stress-related cis-acting elements were found in CarNAC4 promoter, such like NAC recognition site, MYC and MYB recognition site, DRE, LTRE, PRE and ABRE. CarNAC5 promoter contained NAC recognition site, MYC and MYB recognition site, W box, ABRE, GARE and EEC. These finding suggested the cis-acting elements type, amount and location were different between two promoter, which implied CarNAC4 and CarNAC5 could be reponsed to stress and hormone signals in different pathway.3. Electrophoretic mobility shift assays (EMSA) indicated that CarNAC4 and CarNAC5 could specially bind with normal NACRS cis-element while they were not able to bind with mutant NACRS cis-element. Yeast-one-hybrid also showed CarNAC4 and CarNAC5 could specially bind with normal NACRS cis-element while they were not able to bind with mutant NACRS cis-element. The above results proved that CarNAC4 and CarNAC5 proteins had DNA-binding specificity both in vitro and vivo.4. Transgenic Arabidopsis seeds expressing CarNAC4 gene showed improved germination potential under drought and salt condition compared to wild-type seeds. After drought and salt treatments, most wild-type plants were dead and CarNAC4 transgenic plants showed higher survival rate. CarNAC4 transgenic plants also showed higher proline content after drought treatment and lower MDA content after salt treatment. Addtionally, the detached leaves from transgenic plants exhibited significantly lower water loss rate than those of wild-type plants. Furthermore, the expression level of six stress-related genes (RD29A, ERD10, COR15A, COR47, KIN1 and DREB2A) were significantly induced in CarNAC4 transgenic plants under stress condition. These findings indicated over-expression of CarNAC4 gene enhanced tolerance to drought and salt stresses in Arabidopsis Since over-expression of CarNAC4 gene did not affect plant development, it has the potential value for crop drought-resistant genetic engineering.5. Transgenic Arabidopsis seeds expressing CarNAC5 gene showed improved germination potential under drought condition compared to wild-type seeds. After drought treatment, most wild-type plants were dead and CarNAC5 transgenic plants showed higher survival rate. CarNAC4 transgenic plants also showed higher proline content after drought treatment. Addtionally, the detached leaves from transgenic plants exhibited significantly lower water loss rate than those of wild-type plants. Furthermore, the expression level of five stress-related genes (RD22, RD29A, ERD10, COR15A and DREB2A) were significantly induced in CarNAC5 transgenic plants under stress condition. These findings indicated over-expression of CarNAC5 gene enhanced tolerance to drought stresse in Arabidopsis. Like CarNAC4, CarNAC5 also has the potential value for crop drought-resistant genetic engineering.6. Transgenic Arabidopsis plants expressing CarNAC2 gene showed a phenotype of growth retardation such as germination delay, shorter hypocotyls length and later bolting and flowering in comparison with wild-type plants. After drought treatment, CarNAC2 transgenic plants showed higher survival rate than wild-type plants. CarNAC2 transgenic plants also showed higher proline content after drought treatment. Addtionally, the detached leaves from transgenic plants exhibited significantly lower water loss rate than those of wild-type plants. Furthermore, the expression level of four stress-related genes (RD22, RD29A, COR15A and KIN1) were significantly induced in CarNAC2 transgenic plants under drought condition. These findings indicated over-expression of CarNAC2 could enhance drought tolerance and delay reproductive growth, but didn’t decrease the total biomass of plants in Arabidopsis. Therefore, CarNAC2 may be used as a candidate gene for drought-resistance engineering of crop plants, which are expected to provide much vegetative organs.This study have clarified elementarily molecular mechanisms which 3 Chickpea NAC genes responsed to abotic stress through analysis of expression pattern, promoter structure, binding ability and function of transgenic Arabidopsis. All the three genes showed the potential value for crop stress-resistant genetic engineering. |
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