蔗糖转运蛋白VvSUC11和VvSUC12累加作用对提高转基因甜菜含糖量的影响

doi:10.3864/j.issn.0578-1752.2014.12.018

摘要/Abstract

摘要： 【目的】甜菜是重要的糖料作物，块根中的蔗糖含量是其品质的决定因子。利用基因工程技术将葡萄蔗糖转运蛋白基因（VvSUC11和VvSUC12）导入甜菜块根中，以了解蔗糖转运蛋白是否能提高甜菜的含糖量。【方法】利用葡萄蔗糖转运蛋白基因VvSUC11和VvSUC12构建的根特异性表达植物双价表达载体（pCAMBIA2301-SP1-VvSUC11-SP2-VvSUC12），通过农杆菌介导法转化到甜菜（Beta vulgaris L.）品种KWS-9103中。利用PCR和RT-PCR技术检测目的基因是否整合到甜菜中并表达。将获得的转基因甜菜和对照甜菜生根后移栽大田，对其叶片性状、相关的生理指标、块根重和含糖量进行测定。【结果】通过PCR和RT-PCR检测，获得13株转基因甜菜。测得转基因植株叶长、叶宽、叶柄长和叶片数量平均分别为25.16 cm、19.31 cm、29.17 cm和38.33个，与对照相比，叶宽增加31.81%，叶柄缩短16.61%，叶片数目增加17.04%，都存在极显著差异，而叶长增加1.68%，无显著差异；叶绿素a含量（1.31 mg•g-1）、叶绿素b含量（0.562 mg•g-1）和叶绿素总含量（1.87 mg•g-1）与对照无显著差异；叶中可溶性糖含量（14.59 mg•g-1）降低13.04%，与对照（16.51 mg•g-1）存在极显著差异，叶柄中可溶性糖含量（21.90 mg•g-1）增加3.36%，但与对照（21.6 mg•g-1）差异不显著；单株转基因甜菜的块根重和含糖量分别平均为3.067 kg和183.2 g•kg-1，与对照（2.894 kg）相比，块根增重5.91%，差异不显著，含糖量增加9.41%，与对照（167.5 g•kg-1）存在显著差异。说明转基因甜菜叶面积和叶片数的增加，扩大了其光合叶面积，同时叶柄缩短有利于提高糖分子从源叶到库的转运，促进甜菜块根的形成和加快糖分的积累。【结论】利用蔗糖转运蛋白VvSUC11、VvSUC12能够有效地提高转基因甜菜的含糖量。

关键词: 甜菜 , 蔗糖转运蛋白 , 转化 , 农艺性状 , 含糖量

Abstract: 【Objective】Sugar beet is an important sugar crop in the world. The sucrose content in roots is the key determinant of sugar beet quality. In order to study the improvement of saccharinity of sugar beet by two sucrose transporter genes from grape, VvSUC11 and VvSUC12, were introduced into sugar beet root using plant genetic engineering. The work will lay a foundation for using sucrose transporter genes to increase crop yield and quality. 【Method】 A bivalent root specific expression vector called pCAMBIA2301-SP1-VvSUC11-SP2-VvSUC12 containing grape sucrose transporters VvSUC11 and VvSUC12 genes was constructed and transformed into sugar beet variety KWS-9103 by Agrobacterium tumefaciens mediated transformation. Then PCR and RT-PCR analyses were used to confirm the integration and expression of the two genes in transgenic sugar beets. The leaf characters, physiological targets, tuberous root weight and saccharinity of transgenic and control plants were measured after transplanting to the field.【Result】Thirteen transgenic sugar beet lines were obtained by PCR and RT-PCR analyses. Results indicated that the average number of leaf width, petiole length and leaf number of transgenic plants were 19.31 cm, 29.17 cm and 38.33, which increased obviously by 31.81%, 16.61% and, 17.04%, respectively, the average number of leaf length were 25.16 cm, which increased insignificantly by 1.68%. However, there were no significant difference in the content of Chlorophyll a（1.31 mg•g-1）, Chlorophyll b（0.562 mg•g-1） and total Chlorophyll（1.87 mg•g-1） between transgenic and control plants. Compared to control, soluble sugar content (14.59 mg•g-1) of leaf was reduced markedly by 13.04%, but soluble sugar content (21.90 mg•g-1) of petiole was only increased by 3.36%. The average root weight and sugar content of individual plants were 3.067 kg and 183.2 g•kg-1, respectively. Compared with control plants, the root weight of transgenic plants increased by 5.91%, while saccharinity increased significantly by 9.41%. These results demonstrated that larger leaves and higher leaf number lead to the expansion of photosynthetic leaf area, and shorter petiole in transgenic sugar beets was not only helpful to accelerate the rate of transport of sugar molecules from the source leaves to the sink, but also promotes the formation of tuberous root and the accumulation of sugar.【Conclusion】The sugar transporters VvSUC11 and VvSUC12 genes transformation could effectively improve saccharinity in transgenic sugar beet.

Key words: Beta vulgaris , sucrose transporters , transformation , agronomic character , saccharinity

闫甜甜, 郭新勇, 向本春, 祝建波. 蔗糖转运蛋白VvSUC11和VvSUC12累加作用对提高转基因甜菜含糖量的影响[J]. 中国农业科学, 2014, 47(12): 2455-2464.

YAN Tian-Tian, GUO Xin-Yong, XIANG Ben-Chun, ZHU Jian-Bo. Improvement of the Saccharinity by Additive Action of Sucrose Transporters VvSUC11 and VvSUC12 in Transgenic Sugar Beet[J]. Scientia Agricultura Sinica, 2014, 47(12): 2455-2464.

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参考文献

[1]张翼飞, 张晓旭, 刘洋, 徐影, 洪鑫, 李彩凤. 中国甜菜产业发展趋势. 黑龙江农业科学, 2013, 8: 156-160.

Zhang Y F, Zhang X X, Liu Y, Xu Y, Hong X, Li C F. Prospect of sugar beet industry in China. Heilongjiang Agricultural Sciences, 2013, 8: 156-160. (in Chinese)

[2]戚继艳, 阳江华, 唐朝荣. 植物蔗糖转运蛋白的基因与功能. 植物学通报, 2007, 24(4): 532-543.

Qi J Y, Yang J H, Tang C R. Sucrose transporters genes and their functions in plants. Chinese Bulletin of Botany, 2007, 24(4): 532-543. (in Chinese)

[3]Riesmeier J W, Willmitzer L, Frommer W B. Isolation and characterization of a sucrose carrier cDNA from spinach by functional expression in yeast. The EMBO Journal, 1992, 11(13): 4705-4713.

[4]Aoki N, Hirose T, Scofield G N, Whitfeld P R, Furbank R T. The sucrose transporter gene family in rice. Plant and Cell Physiology, 2003, 44(3): 223-232.

[5]Shakya R, Sturm A. Characterization of source and sink-specific sucrose/H+ symporters from carrot. Plant Physiology, 1998, 118(4): 1473-1480.

[6]Noiraud N, Delrot S, Lemoine R. The sucrose transporter of celery. Identification and expression during salt stress. Plant Physiology, 2000, 122(4): 1447-1456.

[7]Dusotoit-Coucaud A, Brunel N, Kongsawadworakul P, Viboonjun U, Lacointe A , Julien J L, Sakr S. Sucrose importation into laticifers of Hevea brasiliensis, in relation to ethylene stimulation of latex production. Annals of Botany, 2009, 104(4): 635-647.

[8]Initiative A G. Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature, 2000, 408(6814): 796-815.

[9]Kühn C, Hajirezaei M R, Fernie A R, Roessner-Tunali U, Czechowski T, Hirner B, Frommer W B. The sucrose transporter StSUT1 localizes to sieve elements in potato tuber phloem and influences tuber physiology and development. Plant Physiology, 2003, 131(1): 102-113.

[10]Wright K M, Roberts A G, Martens H J, Sauer N, Oparka K J. Structural and functional vein maturation in developing tobacco leaves in relation to AtSUC2 promoter activity. Plant Physiology, 2003, 131(4): 1555-1565.

[11]Dusotoit-Coucaud A, Kongsawadworakul P, Maurousset L, Viboonjun U, Brunel N, Pujade-Renaud V, Sakr S. Ethylene stimulation of latex yield depends on the expression of a sucrose transporter (HbSUT1B) in rubber tree (Hevea brasiliensis). Tree Physiology, 2010, 30(12): 1586-1598.

[12]Davies C, Wolf T, Robinson S P. Three putative sucrose transporters are differentially expressed in grapevine tissues. Plant Science, 1999, 147(2): 93-100.

[13]Manning K, Davies C, Bowen H, White P L. Functional characterization of two ripening-related sucrose transporters from grape berries. Annals of Botany, 2001, 87(1): 125-129.

[14]Zhang Y L, Meng Q Y, Zhu H L. Functional characterization of a LAHC sucrose transporter isolated from grape berries in yeast. Plant Growth Regulation, 2008, 54(1): 71-79.

[15]Hattori T, Nakamura K. Genes coding for the major tuberous root protein of sweet potato: Identification of putative regulatory sequence in the 5′upstream region. Plant Molecular Biology, 1988, 11(4): 417-426.

[16]殷东林, 祝建波, 王爱英, 向本春. 蔗糖转运蛋白基因 VvSUC11 和 VvSUC12 双价植物表达载体的构建及在甜菜中的遗传转化. 生物工程学报, 2011, 27(8): 1164-1173.

Yin D L, Zhu J B, Wang A Y, Xiang B C. Construction of a bivalent plant expression vector carrying VvSUC11 and VvSUC12 genes and its genetic transformation in sugar beet. Chinese Journal of Biotechnology, 2011, 27(8): 1164-1173. (in Chinese)

[17]刘巧红, 江莉萍, 于歆. 甜菜叶片组织快速制备DNA的方法. 中国甜菜糖业, 2002, 2: 9.

Liu Q H, Jiang L P, Yu X. A approaching of total DNA isolation from sugarbeet leaves tissue. China Beet and Sugar, 2002, 2: 9. (in Chinese)

[18]冯利平, 韩学信. 芝麻、甘薯、甜菜叶面积校正系数. 山西农业科学, 1992, 5: 20.

Feng L P, Han X X. Leaf area correction factor of sweet Sesame, potato and sugar beet. Journal of Shanxi Agricultural Sciences, 1992, 5: 20. (in Chinese)

[19]白宝璋. 甜菜叶绿素含量的快速测定. 中国糖料, 1987, 2: 29-30.

Bai B Z. Rapid determination of sugar beet chlorophyll content. Sugar Crops of China, 1987, 2: 29-30. (in Chinese)

[20]周祖富, 黎兆安. 植物生理学实验指导. 北京: 中国农业出版社, 2005.

Zhou Z F, Li Z A. Plant Physiology Experiment Instruction. Beijing: China Agriculture Press, 2005. (in Chinese)

[21]杨继春. 自制纸筒育苗移栽技术实施中应着重注意的几个问题. 中国甜菜糖业, 1993, 2: 31-33.

Yang J C. Several problems needed attention in transplanting technology of homemade paper tube seedling. China Beet and Sugar, 1993, 2: 31-33. (in Chinese)

[22]Farrar J, Pollock C, Gallagher J. Sucrose and the integration of metabolism in vascular plants. Plant Science, 2000, 154(1): 1-11.

[23]Williams L E, Lemoine R, Sauer N. Sugar transporters in higher plants–a diversity of roles and complex regulation. Trends in Plant Science, 2000, 5(7): 283-290.

[24]Barker L, Kühn C, Weise A, Schulz A, Gebhardt C, Hiener B, Hellmann H, Schulze W, Ward J W, Frommer W B. SUT2, a putative sucrose sensor in sieve elements. The Plant Cell Online, 2000, 12(7): 1153-1164.

[25]于海彬, 杨文华. 甜菜中的蔗糖运转积累机制. 中国甜菜糖业, 2004, 4: 31-34.

Yu H B, Yang W H. Sucrose accumulation operation mechanism in the sugar beet. China Beet and Sugar, 2004, 4: 31-34. (in Chinese)

[26]Fieuw S, Willenbrink J. Sucrose synthase and sucrose phosphate synthase in sugar beet plants (Beta vulgaris L.). Journal of Plant Physiology, 1987, 131(1): 153-162.

[27]赵宏伟, 邹德堂. 关于甜菜叶片生长特性的研究. 中国糖料, 1997, 4: 16-19.

Zhao H W, Zou D T. Studies on the growth character of sugar beet blade. Sugar Crops of China, 1997, 4: 16-19. (in Chinese)

[28]刘彦琴, 邵金旺, 张家骅. 甜菜体内蔗糖的合成及运输的研究经历和现状. 中国糖料, 1987, 2: 52-56.

Liu Y Q, Shao J W, Zhang J H. Research status of sucrose synthesis and transport in the sugar beet. Sugar Crops of China, 1987, 2: 52-56. (in Chinese)

[29]Gottwald J R, Krysan P J, Young J C, Evert R F, Sussman M R. Genetic evidence for the in planta role of phloem-specific plasma membrane sucrose transporters. Proceedings of the Nationnal Acadmy of Science of the USA, 2000, (25): 13979-13984.

[30]Giaquinta R T. Sucrose translocation and storage in the sugar beet. Plant Physiology, 1979, 63(5): 828-832.

[31]Weise A, Barker L, Kühn C, Lalonde S, Buschmann H, Frommer W B, Ward J M. A new subfamily of sucrose transporters, SUT4, with low affinity/high capacity localized in enucleate sieve elements of plants. The Plant Cell Online, 2000, 12(8): 1345-1355.

[32]Schulze W, Weise A, Frommer W B, Ward J M. Function of the cytosolic N-terminus of sucrose transporter AtSUT2 in substrate affinity. FEBS Letters, 2000, 485(2): 189-194.

[33]Meyer S, Melzer M, Truernit E, HuÈmmer C, Besenbeck R, Stadler R, Sauer N. AtSUC3, A gene encoding a new Arabidopsis sucrose transporter, is expressed in cells adjacent to the vascular tissue and in a carpel cell layer. The Plant Journal, 2000, 24(6): 869-882.

[34]刘宝辉. 玉米蔗糖磷酸合成酶(SPS)基因的克隆及对甜菜遗传转化的研究[D]. 哈尔滨: 东北农业大学, 2003.

Liu B H. Cloning of maize sucrose phophate synthesis gene and transformation into sugar beet [D]. Harbin: Northeast Agricultural University, 2003. (in Chinese)