Cloning and Sequencing Sucrose Synthase Gene of Alpine Potato
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摘要:
目的 蔗糖合酶是植物蔗糖代谢途径中的关键酶，在植物的生长发育过程中具有重要作用。分析蔗糖合酶的核酸序列信息，预测其蛋白结构与功能，以期揭示该酶生物学功能。 方法 通过怀玉山高山马铃薯（Solanum tuberosum L. cv. Huaiyushan，缩写S. tuberosum L. cv. Huaiyushan）试管苗转录组数据库筛选到蔗糖合酶基因的核心片段（PGSC0003DMG400002895，SuSy 4），利用RT-PCR 技术克隆怀玉山高山马铃薯蔗糖合酶基因，并采用生物信息学方法进行序列分析。 结果 怀玉山高山马铃薯蔗糖合酶基因cDNA总长度为2 418 bp，G+C 含量为45.08%；怀玉山高山马铃薯蔗糖合酶由805个氨基酸组成，分子量92 471.33 Da，等电点5.87，为亲水性蛋白；怀玉山高山马铃薯蔗糖合酶的二级结构包括α-螺旋（45.84%）、β -片层（15.16%）、无规则卷曲（39.01%），C端和N端含β -片层和α-螺旋，而无规则卷曲、延伸链、β -片层和α-螺旋则散布于整个蛋白质中；怀玉山高山马铃薯蔗糖合酶的三级结构为四聚体；怀玉山高山马铃薯蔗糖合酶主要存在细胞质、线粒体和叶绿体中；怀玉山高山马铃薯与番茄（Solanum lycopersicum）、潘那利番茄（Solanum pennellii）、智利番茄（Solanum chilense）、马铃薯（Solanum tuberosum）、辣椒（Capsicum annuum）、风铃辣椒（Capsicum baccatum）等6种植物在一个大分支下，这说明怀玉山高山马铃薯蔗糖合酶基因在进化上与这6种植物的亲缘关系较近，尤其是与马铃薯的进化上具有最高的亲缘关系。 结论 怀玉山高山马铃薯蔗糖合酶基因具有典型蔗糖合酶的结构特征，氨基酸序列及核酸序列与同源物种相似度高，在进化上高度保守，对进一步揭示该酶生物学功能具有重要意义。 Abstract:Objective Sequence of the gene associated with the key enzyme in sucrose metabolism pathway of potatoes, sucrose synthase (SuSy), was determined for studying the biological function of the enzyme. Method The core fragment (PGSC0003DMG400002895, SuSy 4) of SuSy was screened from the transcriptome database on the plantlets of Alpine potatoes (Solanum tuberosum L. cv. Huaiyushan). The gene was cloned by RT-PCR, and its sequence analyzed using the bioinformatics method. Result The total length of cDNA of SuSy gene was 2 418 bp containing 45.08% of G+C. The hydrophilic protein had 805 amino acids with a molecular weight of 92 471.33 Da and an isoelectric point of 5.87. Its secondary structure was 45.84% of alpha helix, 15.16% of β -extended strands, and 39.01% of random coils. The β-lamellae and α-helix were found throughout the entire protein but appeared as irregular, curl, extension chains at the C- and N-terminals. The tetrameric gene mainly existed in the cytoplasm, mitochondria, and chloroplast. It closely related to those of S. lycopersicum, S. pennellii, S. chilense, S. tuberosum, Capsicum annuum, and C. baccatum in evolution, especially, S. tuberosum. Conclusion The SuSy gene in the Alpine potato of Huaiyushan had the characteristic structure of a typical sucrose synthase. The amino acid and nucleic acid sequences were highly conserved and homologous to the similar species. The findings would be of great significance for further understanding on the biological function of the enzyme. -
Key words:
- Alpine potato of Huaiyushan /
- plantlet /
- sucrose synthase /
- cloning /
- sequence analysis
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图 1 怀玉山高山马铃薯总RNA的电泳
Figure 1. Electrophoresis of total RNA from Alpine potato of Huaiyushan
图 2 怀玉山高山马铃薯蔗糖合酶基因PCR扩增
Figure 2. PCR amplification of SuSy gene in Alpine potato of Huaiyushan
图 3 怀玉山高山马铃薯蔗糖合酶基因碱基组成
Figure 3. Base composition of SuSy gene in Alpine potato of Huaiyushan
图 4 怀玉山高山马铃薯蔗糖合酶蛋白氨基酸序列
Figure 4. Amino acid sequence of SuSy in Alpine potato of Huaiyushan
图 5 怀玉山高山马铃薯蔗糖合酶蛋白亲疏水值分布
Figure 5. Distribution of relative and sparse water values of SuSy in Alpine potato of Huaiyushan
图 6 怀玉山高山马铃薯蔗糖合酶二级结构
注：大写字母代表氨基酸序列；小写字母h代表α-螺旋（Alpha helix），小写字母e代表延伸链（Extended strand），小写字母c代表无规则卷曲（Random coil）。
Figure 6. Secondary structure of SuSy in Alpine potato of Huaiyushan
Note: the capital letter represents the amino acid sequence; the small letter H represents the alpha helix, the small letter E represents the extended strand, and the small letter C represents the random coil.
图 7 怀玉山高山马铃薯蔗糖合酶二级结构各部分的比例
注：蓝色代表α-螺旋；红色代表延伸链；紫色代表无规则卷曲。
Figure 7. Proportion of various parts in secondary structure of SuSy in Alpine potato of Huaiyushan
Note: Blue represents α-helix; red, extended strand; and, purple, random coil.
图 8 怀玉山高山马铃薯蔗糖合酶三级结构
Figure 8. Tertiary structure of SuSy in Alpine potato in Huaiyushan
图 9 怀玉山高山马铃薯蔗糖合酶系统进化分析
Figure 9. Phylogenetic analysis of SuSy in Alpine potato of Huaiyushan
表 1 怀玉山高山马铃薯蔗糖合酶蛋白各氨基酸的数目和比例
Table 1. Number and proportion of amino acids of SuSy in Alpine potato of Huaiyushan
氨基酸种类
Types of amino acids数目
Number比例
Proportion/%Ala（A） 56 7.0 Arg（R） 44 5.5 Asn（N） 30 3.7 Asp（D） 39 4.8 Cys（C） 7 0.9 Gln（Q） 22 2.7 Glu（E） 75 9.3 Gly（G） 46 5.7 His（H） 28 3.5 Ile（I） 48 6.0 Leu（L） 89 11.1 Lys（K） 51 6.3 Met（M） 22 2.7 Phe（F） 41 5.1 Pro（P） 35 4.3 Ser（S） 36 4.5 Thr（T） 41 5.1 Trp（W） 11 1.4 Tyr（Y） 31 3.9 Val（V） 53 6.6 
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