摘 要

TMM基因是最早被发现影响气孔发育的基因之一，在植物气孔发育途径中，主要作为负调控者，通过抑制不对称分裂以控制气孔的正常分布。TMM基因编码一个富含亮氨酸重复序列的受体类蛋白(LRR-RLP)，蛋白的保守区域是亮氨酸重复序列区域(LRR），它包含10个连续的植物类型亮氨酸重复序列，与拟南芥中的受体类激酶(LRR-RLP)家族是相关的，但与其不同的是，TMM蛋白缺少一个细胞内的激酶区域，这种结构表明，TMM蛋白有可能与其他有具有激酶区域的蛋白形成受体复合体以调控气孔发育。

本实验选择对小叶杨气孔发育关键基因为TMM切入点，克隆其ORF阅读框全长，并利用生物信息学软件对其特性进行预测分析，连接载体并转化大肠杆菌，筛选后再进行菌落PCR阳性检测。将已获得的TMM序列进行电子拼接后得到基因的全长cDNA序列。用NCBI网站进行序列分析，可知：核苷酸序列的可读框全长为1347bp，编码了449个氨基酸的阅读框，含起始密码子(ATG)和终止密码子(TAA)。利用生物信息学软件对蛋白质理化参数进行分析。分析结果为：TMM基因编码的氨基酸共有448个，分子量约为48887.11，蛋白质的等电点为7.67。然后利用相关软件对序列进行了蛋白质的疏水性，信号肽，跨膜区域，以及蛋白质高级结构的预测等分析。本研究为探讨小叶杨气孔发育关键基因为TMM在气孔发育中的分子机制奠定基础，对进而小叶杨抗旱耐盐性能机理进行较为深入和系统的研究，具有一定的理论意义。

关键词：TMM基因；小叶杨；气孔

Gene clone and specificity analysis of TMM gene of Populus simonii

ABSTRACT

The TMM gene is one of the earliest genes that have been found to affect stomatal development. It is mainly a negative regulator in the way of stomatal development in plants to control the normal distribution of stomata by inhibiting asymmetric division. The TMM gene encodes a receptor class protein (LRR-RLP) rich in leucine repeat sequence. The conserved region of the protein is the leucine repeat sequence region (LRR), which contains 10 continuous plant type leucine repeat sequences related to the receptor like kinase (LRR-RLP) family in Arabidopsis, but is different from the TMM protein deficiency. There is a few intracellular kinase regions, which suggest that the TMM protein may form a receptor complex with other kinase regions to regulate stomatal development.

In this experiment, the key base of stomatal development of poplar was selected as the TMM entry point, and the whole length of the ORF reading frame was cloned. The characteristics were predicted and analyzed by bioinformatics software, the vector was connected and the Escherichia coli was transformed, and the positive detection of colony PCR was carried out after screening. The full-length cDNA sequence of the gene was obtained by electronic splicing of the obtained TMM sequence. The sequence analysis of NCBI website shows that the readable frame of the nucleotide sequence is 1347bp, which encodes a reading frame of 449 amino acids, including the starting codon (ATG) and the terminating codon (TAA). The physicochemical parameters of protein were analyzed by bioinformatics software. The results showed that the TMM gene encoded 448 amino acids with a molecular weight of about 48887.11 and an isoelectric point of 7.67. Then, we analyzed the hydrophobicity, signal peptide, transmembrane domain, and protein structure prediction of the protein sequence. In this study, the key basis of the stomatal development of poplar poplar is to lay the foundation for the molecular mechanism of TMM in the development of stomata. It is of certain theoretical significance to further and systematically study the mechanism of drought and salt tolerance of poplar.

Key words：TMM gene；Simon poplar；Stoma

目 录

1 文献综述………………………………………………………………………………………1

1.1 气孔………………………………………………………………………………………1

1.1.1 气孔的简介……………………………………………………………………………1

1.1.2 气孔的发生过程………………………………………………………………………1

1.1.3 影响气孔发育的遗传因素……………………………………………………………1

1.1.4 影响气孔发育的环境因素……………………………………………………………1

1.2 TMM基因……………………………………………………………………………………2

1.2.1 TMM基因的发现及命名…………………………………………………………………2

1.2.2 TMM基因的特点…………………………………………………………………………2

1.2.3 TMM基因的作用机制……………………………………………………………………2

1.3 植物基因克隆技术的发展及ORF克隆技术………………………………………………2

1.4 小叶杨…………………………………………………………………………………………3

1.5 本论文的研究目的………………………………………………………………………………4

2 材料与方法………………………………………………………………………………5

2.1 材料…………………………………………………………………………………………5

2.1.1 植物材料……………………………………………………………………………5

2.1.2 载体…………………………………………………………………………………5

2.2 仪器与设备………………………………………………………………………………………5

2.3 实验方法…………………………………………………………………………………………6

2.3.1 获得TMM基因的全长序列………………………………………………………6

2.3.2 总RMA的提取与纯化……………………………………………………………6

2.3.3 反转录反应…………………………………………………………………………7

2.3.4 全长基因的克隆及ORF扩增………………………………………………………7

2.3.5 DNA琼脂糖凝胶电泳………………………………………………………………8

2.3.6 切胶回收……………………………………………………………………………8

2.3.7 连接T-Vector…………………………………………………………………………9

2.3.8 大肠杆菌转化………………………………………………………………………10