【作者】 张执金；

【导师】 黄荣峰；

【作者基本信息】 中国农业科学院 ， 生物化学与分子生物学， 2004， 硕士

【摘要】 基因表达调控是植物胁迫应答的重要环节。植物激素和转录因子对植物胁迫应答中的基因表达调控起着关键性的作用。ERF类转录因子是植物特有的一个转录因子大家族，它们都具有一个保守的DNA结合域—ERF domain。ERF类转录因子可调节植物对病原物、低温、干旱、高盐及伤害等生物与非生物胁迫的适应性应答，在植物胁迫应答中起着重要的作用。乙烯是植物体内一种多功能植物激素。早期研究表明，在植物的生物胁迫应答中乙烯可以通过ERF转录因子调节大量含有GCC-box(TAGCCGCC)的PR基因表达，提高植物对生物胁迫的抗／耐性，在植物的生物胁迫应答中发挥着重要的作用。近几年的研究表明乙烯也可以通过ERF转录因子参与植物的非生物胁迫应答，提高植物的非生物胁迫耐性。本实验室以GCC-box为bait，利用酵母单杂交系统从番茄cDNA表达文库中筛选到两个新基因LeERF2、JERF2。研究表明它们都属于ERF类转录因子，并参与乙烯和植物的非生物胁迫应答。本实验是在我们实验室原有的研究基础上进一步研究了LeERF2、JERF2在植物非生物胁迫应答中的功能，并分析它们可能参与的调控途径。 凝胶阻滞实验(又称EMSA)结果表明LeERF2蛋白具有结合GCC-box和DRE／CRT两种顺式作用元件的能力，但不能同ABRE偶联元件CE1结合。转LeERF2基因烟草的Northern杂交结果表明LeERF2的超表达除了导致转基因烟草中含有GCC-box的基因osmotin和PRB-1b的组成型表达外，某些含有DRE／CRT顺式作用元件的基因COR15a和RD17也组成型表达。但另外一些含有DRE／CRT的基因如RD29A和含有ABRE的基因RD29B的表达则不受影响。分别用NaCl、甘露醇和葡萄糖处理野生型和LeERF2转基因烟草种子时发现LeERF2提高了转基因烟草种子的胁迫耐性。低温处理转基因烟草时，LeERF2提高了转基因烟草植株的耐冻性。用-6℃处理烟草植株八小时并恢复生长两天，野生型烟草的死亡率达到86.7％，而转基因植株仅为17％。此外LeERF2还提高转基因烟草幼苗根的盐胁迫耐性。这些结果表明LeERF2基因可能通过调节某些含有GCC-box或DRE／CRT顺式作用元件的基因的表达参与植物的非生物胁迫应答，提高了植物的非生物胁迫耐性。 对JERF2的研究表明JERF2提高了转基因烟草种子对NaCl和甘露醇的胁迫耐性，而ABA处理转基因烟草种子时发现ABA对JERF2的功能没有明显的影响。对转基因烟草植株的研究表明JERF2提高了转基因植株提对PEG处理下的渗透胁迫的耐性，也显著提高了转基因烟草对NaCl的胁迫耐性。转基因烟草的Northern杂交结果表明LeERF2的超表达可造成某些含有GCC-box的基因如osmotin和PRB-1b的组成型表达，但对含有DRE／CRT顺式作用元件的基因COR15a、RD17和RD29A及含有ABRE顺式作用元件的基因RD29B的表达没有影响。这些结果表明JERF2可能通过调节某些含有GCC-box的基因的表达而提高了植物的非生物胁迫耐性。另外JERF2转基因烟草的乙烯三重反应实验表明JERF2还可能影响乙烯的生成，参与植物体内的其它功能。以上实验结果证明LeERF2、JERF2可以通过不同途径调节植物胁迫相关基因表达，参与植物非生物胁迫应答，提高植物对非生物胁迫的耐性。表明了ERF类转录因子在植物胁迫应答中具有重要作用。更多还原

【Abstract】 It is the vital step for the response to the stresses of plants to regulate the gene expressions. Hormones and transcription factors play an important role on regulating the gene expressions of plants. ERF transcription factors, which contain a conservative DNA binding domain-ERF domain, and regulate the responses to both biotic and abiotic stresses of plants, such as disease, low temperature, drought, high salinity and wound, belong to a large transcription factors family , which is unique to plants. The ERF transcription factors play a key role on the responses to various stresses of plants. Ethylene (ET) is an important hormone, which can produce a myriad of effects on plant. The early studies showed that ET is important for plants to respond to biotic stress by regulating the expressions of numerous PR genes containing the GCC-box (TAGCCGCC) through ERF transcription factors. In Recent several years, studies supported the fact that ET exerts significant effects on the response to abiotic stress of plants through ERF transcription factors. Our label screened the cDNA expressive library of tomato by yeast one-hybrid system with the GCC-box bait and got two new genes: LeERF2 and JERF2. Studies showed that both of them belong to the ERF transcription factors family. This study is to analyze the functions and regulatory pathway of LeERF2 and JERF2 in the responses to abiotic stresses of plants on the base of the former studies of our label.Our studies illustrated the LeERF2 protein can bind to both GCC-box and DRE/CRT cis-elemengs, but fail to ABRE coupling element (CE1) in the EMSA. We also found that LeERF2 can result in the constitutive expressions of genes containing either GCC-box or DRE/CRT cis-elemengs in their promoters, such as osmotin, PRB-lb, COR15a and RD17 in transgenic tobacco lines. But LeERF2 had no effect on the expression of RD29A or RD29B which containing ABRE element in their promoters. LeERF2 obviously enhanced the tolerance of transgenic tobacco seeds overexpressing the LeERF2 under the salinity and osmotic stresses by treated with NaCl, mannitol, glucose, respectively. LeERF2 transgenic tobacco also demonstrated highly freezing tolerance under the low-temperature treatment. For example, the death rate of wild type is over 80% under the stress of freezing at -6 C for 8 hours, whereas, the transgenic line is only 17%. Another, LeERF2 enhanced the tolerance of salt stress of root of transgenic tobacco lines. From the above results, we believed that LeERF2 enhanced the abiotic tolerance of plants by regulating the expression of genes encoding GCC-box or DRE/CRT element in their promoter.JERF2 also enhanced the tolerance of abiotic stress of transgenic seeds under the stresses of NaCl and mannitol. JERF2 increased the tolerance of transgenic tobacco plants under the stress of NaCl and PEG treatment. Whereas the results of the Northern blotting experiment showed that JERF2 only regulated the expressions of genes osmotin and PRB-lb containing GCC-box cis-elemengs in their promoters in the transgenic tobacco plants, but failed to effect the expressions of COR15a, RD17 RD29A and RD29B containing DRE/CRT or ABRE element in their promoters. Another, we found that transgenic tobacco demonstrated the constitutivephenotype of ethylene for the effect on the production of ET.All above showed that LeERF2, JERF2 perform key function in the response to abiotic stresses of plants and can enhance the tolerance of plants through the different signaling pathways.更多还原