Cloning and function analysis of starch synthase SSⅡa promoter in maize
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摘要:目的
克隆玉米Zea mays淀粉合成酶SSⅡa启动子，并分析其功能，为进一步研究和应用SSⅡa启动子奠定基础。
方法通过NCBI上公布的玉米基因组序列，在网站MaizeGDB上BLAST查找到SSⅡa 5′侧翼序列，利用PCR方法从玉米B73中克隆SSⅡa启动子；通过PlantCare在线分析启动子顺式作用元件，用特异性引物分别克隆出长度为1 407、867、633、483和365 bp的片段，与植物表达载体pCAMBIA3301连接，构建5种5′缺失体的植物表达载体，命名为P1、P2、P3、P4和P5。用农杆菌介导法转化拟南芥Arabidopsis thaliana，获得转基因拟南芥。
结果以玉米B73基因组DNA为模板，用特异性引物SSⅡaF/SSⅡaR进行扩增，得到2 526 bp序列；除草剂筛选的阳性拟南芥植株PCR验证均检测出gus基因；GUS组织化学分析表明，5种类型启动子构建的表达载体在成熟期叶片、果荚中均显蓝色；gus基因定量分析表明，成熟期5种转基因拟南芥叶片中， gus基因表达量P1最高，其他基本一致；种子中gus基因表达量P1和P2相近，且高于P3、P4和P5。
结论成功克隆玉米SSⅡa启动子；构建的5种SSⅡa启动子缺失体表达载体在转基因拟南芥中均具有活性，长度为1 407 bp（P1）和867 bp（P2）的启动子具有胚乳特异性。
Abstract:ObjectiveTo clone maize (Zea mays) starch synthase SSⅡa promoter, analyze its function, and provide a basis for its future research and application.
MethodThe SSⅡa 5′ flanking sequence was found on Maize GDB by BLASTing the maize genome sequence published on NCBI, and the SSⅡa promoter was cloned from maize B73 using PCR. We analyzed the cis elements of the promoter using PlantCare. Fragments of 1 407, 867, 633, 483, and 365 bp were cloned with specific primers, and were inserted into the plant expression vector pCAMBIA3301, respectively. Five plant expression vectors with different 5′ deletions of the SSⅡa promoter were constructed and named P1, P2, P3, P4 and P5.The transgenic Arabidopsis thaliana plants were obtained through Agrobacterium-mediated transformation.
ResultA DNA fragment of 2 526 bp was obtained by PCR amplification with maize B73 genome DNA as template and SSⅡaF/SSⅡaR as specific primers. The positive A.thaliana plants, which were screened by herbicide, had gus gene by PCR detection. The histochemical analysis of GUS showed that the expression vectors of five promoters were blue in leaves and pods at maturity. The quantitative analysis of gus gene showed that among five transgenic A.thaliana at maturity, the expression level of P1 in leaves was the highest and the others were basically the same, and the expression levels of P1 and P2 in seeds were similar, both being higher than those of P3, P4 and P5.
ConclusionThe maize SSⅡa promoter has been successfully cloned. The five constructed expression vectors with different 5′ deletions of the SSⅡa promoter all have activities in transgenic A.thaliana, and the promoters with the length of 1 407 bp (P1) and 867 bp (P2) have endosperm specificity.
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Keywords:
- maize /
- starch synthesis enzyme /
- SSⅡa promoter /
- cloning /
- vector construction /
- function analysis
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图 1 SSⅡa启动子克隆和PCR验证的电泳结果
M：DL2000 DNA Marker；1~3：SSⅡa启动子。
Figure 1. Electrophoresis results of SSⅡa promoter cloning and PCR amplification
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图 2 玉米SSⅡa启动子的序列分析
附有黄色底纹的序列为相关顺式作用元件，由上至下红色方框内依次为克隆突变碱基、胚乳特异性表达功能元件、参与抗旱诱导的MYB结合位点、TATA-box、SSⅡa基因起始密码子ATG。
Figure 2. Sequence analysis of maize SSⅡa promoter
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图 3 不同长度SSⅡa启动子片段的扩增、重组载体PCR验证及双酶切验证结果
M：DL2000 DNA Marker；1~5分别为缺失体P1、P2、P3、P4和P5。
Figure 3. Amplification of SSⅡa promoter fragments of different length, PCR verification of recombinant vectors and double enzyme digestion verification
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图 4 不同表达载体gus基因PCR扩增验证电泳结果
M：DL2000 DNA Marker；1~2为缺失体P5，3~4为缺失体P4，5~6为缺失体P3，7~8为缺失体P2，9~10为缺失体P1；CK：非转基因对照。
Figure 4. PCR detection results of gus gene in different expression vectors by electrophoresis
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图 5 成熟期转基因拟南芥植株叶片和果荚GUS组织化学染色
Figure 5. Histochemical analysis of GUS activities in leaves and pods of transgenic Arabidopsis thaliana plant at maturity
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图 6 成熟期转基因拟南芥叶片和种子中gus基因的相对表达量
Figure 6. Relative expression levels of gus gene in leaves and seeds of transgenic Arabidopsis thaliana plant at maturity
表 1 SSⅡa启动子区顺式作用元件
Table 1 Cis-acting elements of SSⅡa promoter
元件名称 序列 元件功能 元件位置 CAAT-box CAATT、CAAAT、CAAT 启动子和增强子区常见的顺式作用元件 -2 380、-1 094、-2 088、-1 165、-1 377、-1 166、-1 095、-1 915、-2 013、-1 032、-815 CAT-box GCCACT 与分生组织表达相关的作用元件 -1 225、-546、-127 CE3 GACGCGTGTC 参与ABA和VP1响应的作用元件 -2 430 CG-motif CCATGGGG 光响应元件 -1 945 G-box CACATGG 光响应元件 -1 601、-392 I-box GGATAAGGTG 光响应元件 -2 419 MBS CGGTCA MYB结合位点 -2 452 Skn-1-motif GTCAT 胚乳表达所需的作用元件 -1 497、-1 087 GCN4-motif CAAGCCA 胚乳表达调控作用元件 -875 Sp1 CC(G/A)CCC 光响应元件 -1 279、-1 145 TATA-box TAATA、TATA、ATATAA、 核心启动子元件（在转录起始位点上游） -1 850、-2 028、-1 389、-1 847、-1 931、-1 848、-2 030、-529、-508 TC-rich repeats ATTTTCTCCA 参与防御和应激反应的作用元件 -1 815 TGA-element AACGAC 生长素响应的作用元件 -1 256、-247 TGACG-motif TGACG 参与茉莉酸甲酯响应的作用元件 -2 458、-1 198、-754 ABRE CACGTG 参与脱落酸响应的元件 -392 CGTCA-motif CGTCA 参与茉莉酸甲酯响应的作用元件 -213 GARE-motif TCTGTTG 赤霉素应答作用元件 -1 029 HSE AAAAAATTTC 参与热胁迫应答的作用元件 -824 MBS CAACTG 参与抗旱诱导的MYB结合位点 -1 000、-761 O2-site GATGATGTGG 参与玉米醇溶蛋白代谢调控的作用元件 -1 044 motif IIb CCGCCGCGCT 脱落酸响应元件 -796 
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