摘要
山梨醇是蔷薇科植物主要的光合产物、运输糖和贮藏物质，起着其他植物中
蔗糖的作用。山梨醇还是一种小分子渗透物质，可以改变细胞渗透势，因而与植物抗性密切相关。另外，果实的味道在很大程度上决定于山梨醇转变成的糖的类型。酶对山梨醇的代谢起关键作用， 6-磷酸山梨醇脱氢酶（S6PDH）是山梨醇合成最重要的一种酶，催化6-磷酸葡萄糖转化为6-磷酸山梨醇，再通过6-磷酸山梨醇磷酸酯酶转变为山梨醇。鉴于此 ，一些学者对S6PDH进行了大量研究，取得了一定进展。本研究通过苹果S6PDH cDNA的基因克隆、序列分析及其植物表达载体的构建，为该基因转化其他非蔷薇科果树奠定重要基础,为进一步研究山梨醇的代谢及提高非蔷薇科果树抗性提供一种新途径。
本试验首先以“皇家嘎拉”苹果叶片为试材，采用三种方法提取了总RNA并加以比较。结果表明三种方法均可提取出高质量的RNA，但三种方法繁琐程度不同，最后选择方法一并稍作改动用于以后的研究。
RT-PCR涉及的因素较多，本研究对RT-PCR体系中各物质量进行了研究，建立了稳定高效的反应体系，获得了符合大小的目的片段。RT体系：dNTPs(2mmol/L)2.5μl；5×Buffer 4μl；RNasin（50U/μl）1μl；Oligo(dT)（10μmol/L）0.5μl；总RNA模板4μl；Mo-MLV Rtase（200U/μl）1μl；DEPC水补足20μl。PCR体系：dNTPs(2mmol/L)5μl；10×Buffer 2.5μl；MgCl2（25mmol/L）2.4μl；LD03（10μmol/L）2μl；LD04（10μmol/L）2μl；Taq 酶（5U/μl）0.3μl； RT产物4μl；DEPC水补足25μl。
本研究进一步对RT-PCR的产物进行了克隆、测序及分析。结果表明该片段全长933bp，编码310个氨基酸。经过序列同源性分析，其与Kanayama等发表的序列有4个核苷酸不同,但由于密码子存在简并性，使得两片段所编码的氨基酸序列只在第四位不同，即在我们研究的片段中为甘氨酸，而在Kanayama等发表的序列中为颉氨酸。同时还对该基因的酶切位点进行了分析。
为了进行以后的研究，本试验借助pUCm-T载体和pGEm-7Zf载体将S6PDH全长cDNA插入到植物表达载体pBI121的多克隆位点上，构建了含有S6PDH全长cDNA的植物表达载体pBIS，并通过直接导入法将其导入农杆菌EHA105。
Sorbitol is the major photosynthetic product、translocating sugar alcohol and storage carbohydrate in Rosaceae plants, the role sorbitol played is like sucrose in other plants. As low-molecular-weight osmotic compounds, it can adjust infiltrative level, so it’s correlated with resistance. In addition, fruit flavor is most dependent on sugar types transformed from sorbitol. Enzyme plays the most important role in sorbitol metabolism. S6PDH is the key enzyme for the biosynthesis of sorbitol, which catalyzed the conversion between glucose-6-phosphate and sorbitol-6-phosphate. So lots of work has done by many researchers, and gain large progress. The aim of the study is to lay foundations for transferring the gene encoding S6PDH, by means of cloning and sequence analysis of S6PDH gene and constructing of the plant expression vector.
At first, the three methods were adopted to extract RNA from apple leaves. The results showed that three methods were all suitable for RNA extraction. The first method is the simplest one.
Second，the factor that affect RT-PCR were studied and highly efficient RT-PCR detection system was established and optimized. As for the 20μl RT system: dNTPs(2mmol/L)2.5μl; 5×Buffer4μl; RNasin（50U/μl）1μl; Oligo(dT)（10μmol/L）0.5μl; template RNA4μl; Mo-MLV Rtase（200U/μl）1μl.. As for the 25μl PCR system: dNTPs(2mmol/L)5μl; 10×Buffer2.5μl; MgCl2（25mmol/L）2.4μl; LD03（10μmol/L）2μl; LD04（10μmol/L）2μl; Taq DNA ploymerase（5U/μl）0.3μl; cDNA 4μl.
Third, the special fragment of RT-PCR was used to cloning and sequence analysis. The cDNA of S6PDH cloned from apple leaves, consisted of 930bp,encoding a protein of 310 amino acids. Obtained sequence was compared with that of Kanayama’s research, there were 4 different bases in 930 nucleotides. There was only one different amino acid due to code degeneracy. Simultaneity, restriction sites analysis was done.
Finally, the plant expression vector pBIS was constructed containing complete S6PDH cDNA via pUCm-T and pGEm-7Zf,and combinant was transferred into agrobacterium tumefacien by direct DNA transfer and the transformant was identified with PCR.
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