Activity analysis of different length fragments of walnut JrTT1-1 promoter in response to drought stress
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摘要:
目的 TTl是C2H2-ZFP（WIP型锌指结构）类转录因子调控蛋白，核桃JrTT1-1基因启动子含有干旱响应元件，具有调控干旱胁迫的功能。本研究通过分离JrTT1-1基因不同长度启动子片段并对其受干旱胁迫后的表达活性进行分析，探讨JrTT1-1基因响应干旱胁迫的机制。 方法 根据WRKY顺式作用元件的分布，将JrTT1-1基因启动子分为1 002 bp（−1 ~ −1 002）、720 bp（−1 ~ −720）、448 bp（−1 ~ −448）、174 bp（−1 ~ −174）、149 bp（−1 ~ −149）5个片段，分别记为S1、S2、S3、S4、S5。用S1、S2、S3、S4、S5分别替换pCAMBIA1301载体的CaMV35S启动子构建重组载体，通过农杆菌介导的蘸花法转化拟南芥，经潮霉素筛选、PCR验证及GUS基因表达分析确定后培养至T3代。对不同生长期不同组织进行GUS酶活性测定，评价不同片段的时空表达活性。将S1、S2、S3、S4、S5转基因植株种子萌发生长30 d进行干旱处理（50 mmol/L甘露醇），未干旱处理的设为对照（CK），分析整株、根及地上部分GUS酶活性，评价不同片段响应干旱的差异。 结果 正常生长条件下，S1、S2、S3、S4、S5转基因拟南芥在不同生长时期、不同组织器官中均能检测出GUS酶活性，但不同片段GUS活性具有差异，且随着片段变短，活性降低；但S1和S2之间的差异不显著。比较成熟种子、鲜种子、35 d根、茎、叶、花的GUS活性，发现不同组织之间也有区别，体现了5个片段的组织表达特异性。与CK相比，干旱胁迫下，5个片段整株、根和地上部分的GUS活性均显著提高，其中干旱胁迫后S1、S2、S3、S4、S5全株的GUS活性分别为CK的1.50、1.46、1.47、1.46、2.23倍，根GUS活性分别为CK的1.29、1.29、1.28、1.53、1.36倍，地上部分GUS活性分别为CK的1.62、1.59、1.57、1.59、2.30倍。 结论 JrTT1-1基因启动子片段的表达活性与其长度呈正相关性，每个长度启动子片段活性具有根、茎、叶、花、种子等组织特异性；WRKY元件及其数量可能与干旱胁迫调节作用相关，且JrTT1-1启动子在干旱胁迫下的表达也具有组织差异性。 Abstract:Objective TT1 is a C2H2-ZFP (WIP-type zinc finger structure) transcription factor regulatory protein. Walnut JrTT1-1 gene promoter contains drought response elements and has the function of regulating drought stress. In this study, the different length promoter fragments of JrTT1-1 gene were isolated and their expression activity after drought stress was analyzed to explore the mechanism of JrTT1-1 gene response to drought stress. Method According to the distribution of WRKY cis-acting elements, the promoter of JrTT1-1 gene was divided into 5 fragments of different lengths: 1 002 bp (−1 − −1 002), 720 bp (−1 − −720) , 448 bp (−1 − −448), 174 bp (−1 − −174) and 149 bp (−1 − −149), and they were denoted as S1, S2, S3, S4, S5, respectively. The recombinant vector was constructed by replacing the CaMV35S promoter of pCAMBIA1301 vector with S1, S2, S3, S4 and S5, and transformed into Arabidopsis thaliana by Agrobacterium-mediated dipping method. The transgenic lines were confirmed by hygromycin screening, PCR verification and GUS gene expression evaluation. Then the transgenic lines were cultivated to T3 generation for further analysis. GUS activity was measured in different tissues at different growth stages to evaluate the temporal and spatial expression activity of different fragments. The seeds of S1, S2, S3, S4, and S5 transgenic plants were germinated and grown to 30-d-old and then subjected to drought treatment (50 mM mannitol), no drought treatment was set as the control (CK), the GUS enzyme activities of the whole plant, roots and aerial parts were analyzed to evaluate the difference of varied fragments in response to drought. Result Under normal growth conditions, GUS activity can be detected in S1, S2, S3, S4, S5 transgenic Arabidopsis in different growth stages and in varied tissues and organs, but the GUS activity of different fragments was different, and the activity decreased as the fragment became shorter; but the difference between S1 and S2 was not significant. Comparing the GUS activity in mature seeds, fresh seeds, 35-day-old roots, stems, leaves, and flowers, it was found that there were differences between different tissues, which reflected the specificity of the tissue expression of five fragments. Under drought stress, the GUS activities of the whole plant, roots and aerial parts of S1, S2, S3, S4, and S5 transgenic plants were significantly increased: the GUS activities of the whole plant were increased by 1.50-, 1.46-, 1.47-, 1.46-, 2.23-fold, the roots were enhanced by 1.29-, 1.29-, 1.28-, 1.53-, 1.36-fold, and the aerial parts were strengthen by 1.62-, 1.59-, 1.57-, 1.59-, 2.30-fold, respectively, of those under normal conditions. Conclusion The expression activity of the JrTT1-1 gene promoter fragment is positively correlated with its length, and the activity of each length promoter fragment is specific to tissues of root, stem, leaf, flower, and seed. WRKY elements and their numbers may be related to the regulation of drought stress, and the expression of JrTT1-1 promoter segments under drought stress also has tissue differences. -
Key words:
- walnut /
- JrTT1-1 gene /
- promoter /
- GUS activity /
- drought stress
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图 1 依据WRKY识别元件划分的5个不同长度启动子片段
Figure 1. Situation of 5 promoter fragments of different length divided according to WRKY recognition motif
图 2 启动子片段插入pCAMBIA1301的重组载体示意图
Figure 2. Schematic diagram of the recombinant vector with the promoter fragment inserted into pCAMBIA1301
图 3 5个片段转入拟南芥各株系的GUS基因表达量
S11 ~ S18、S21 ~ S28、S31 ~ S38、S41 ~ S48、S51 ~ S58分别为转S1、S2、S3、S4、S5片段的8个株系。S11 − S18, S21 − S28, S31 − S38, S41 − S48, S51 − S58 are 8 lines transformed with S1, S2, S3, S4 and S5 fragments, respectively.
Figure 3. GUS gene expression levels of 5 fragments transferred Arabidopsis lines
图 4 不同启动子片段转基因拟南芥株系不同生长期（A）和不同组织（B）的GUS活性
图上小写字母表示同生长阶段或同组织5个启动子片段之间的差异显著性（P < 0.05）。Different lowercase letters above the bars indicate significant difference between 5 promoter segments in the same growth stage or tissue (P < 0.05).
Figure 4. GUS activity in different growth stages (A) and tissues (B) of transgenic Arabidopsis lines with different promoter fragments
图 5 不同启动子片段转基因植株种子萌发不同天数（A）及不同组织（B）GUS活性与片段长度的相关性Fig. 5 Correlations between GUS activity and fragment length of transgenic plants with different promoter fragments for different days of seed germination (A) and different tissues (B)
图 6 不同启动子片段转基因拟南芥株系在干旱胁迫下的GUS活性
A ~ C分别为30 d大植株的整株、根、地上部分的GUS活性。*表示同一个株系在甘露醇处理下的GUS活性与对照比差异显著（P < 0.05）。A−C are the GUS activity of the whole plant, roots and aerial parts of 30-day-old plants, respectively. * indicates that the GUS activity of the same line under mannitol treatment is significantly different from the control (P < 0.05).
Figure 6. GUS activity of transgenic Arabidopsis lines with different promoter fragments under drought stress
表 1 不同片段启动子插入pCAMBIA1301的引物
Table 1. Primers used for different promoter fragments inserted into pCAMBIA1301
片段
Fragment上游引物
Forward primer下游引物
Reverse primerS1 5′-ATGCGGTACCTTCACTGATCAGTACAATAT-3′ 5′-TAGCGGATCCGGTTAAAGATTTGATAGGAAG-3′ S2 5′-ATGCGGTACCAAGTGACTCAAGTTACATAT-3′ S3 5′-ATGCGGTACCTTGACGGTGGCAAGTGAAAAAG-3′ S4 5′-ATGCGGTACCGTCTGCAGGGGGTAATTTCT-3′ S5 5′-ATGCGGTACCTTCCATGCTTGCTTTCAGCT-3′ 注：上游引物中下划线为酶切位点Kpn1，下游引物中下划线为酶切位点BamH1。Notes: underline in the forward primer is Kpn1, and the underline in the reverse primer is BamH1. 
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