齐希梁;李明;刘聪利;宋露露;

• 1:中国农业科学院郑州果树研究所

摘要(Abstract)：

【目的】建立欧洲甜樱桃果实VIGS体系。【方法】利用In-Fusion cloning技术,构建欧洲甜樱桃的烟草脆裂病毒(TRV)介导基因沉默表达载体pTRV2-ANS,转入农杆菌GV3101中,采用注射压迫法侵染欧洲甜樱桃果实,通过分子检测、qRT-PCR和表型观察技术评价TRV介导VIGS沉默体系的效果。【结果】TRV::ANS侵染的欧洲甜樱桃果实ans基因的表达显著低于TRV::00侵染的果实。与TRV::00比较,TRV::ANS侵染的欧洲甜樱桃果实的果皮和果肉颜色都没有发生着色,果皮和果肉颜色呈绿色,而TRV::00侵染的欧洲甜樱桃果实的果皮和果肉正常着色。【结论】研究建立的TRV介导欧洲甜樱桃VIGS体系可有效沉默欧洲甜樱桃果实内源基因表达。该体系为欧洲甜樱桃果实品质基因的功能验证及分子机制研究奠定了技术基础。

关键词(KeyWords)： 欧洲甜樱桃;TRV;VIGS;基因沉默;果实

Abstract:

Keywords:

基金项目(Foundation): 中国农业科学院科技创新工程专项(CAAS-ASTIP-2018-ZFRI);; 中央级科研院所基本科研业务费专项(1610192018201)

作者(Author): 齐希梁;李明;刘聪利;宋露露;

Email:

DOI:

参考文献(References)：

• [1] LI B,XIE Z,ZHANG A,XU W,ZHANG C,LIU Q,LIU C,WANG S. Tree growth characteristics and flower bud differentiation of sweet cherry(Prunus avium L.)under different climate conditions in China[J]. Horticultural Science,2010,37(1):6-13.
• [2]李坤坤,徐昌杰.蔷薇科果树离体再生与遗传转化研究进展[J].园艺学报,2017,44(9):1633-1644.LI Kunkun,XU Changjie. Advances in regeneration and genetic transformation of Rosaceae fruit trees[J]. Acta Horticulturae Sinica,2017,44(9):1633-1644.
• [3]王新平,茹慧玲,孙慧英,王新生,杨兆亮,杨雪瑞.果树转基因研究进展[J].山西农业科学,2016,44(1):123-125.WANG Xinping,RU Huiling,SUN Huiying,WANG Xinsheng,YANG Zhaoliang,YANG Xuerui. Research progress of transgenic fruit trees[J]. Journal of Shanxi Agricultural Sciences,2016,44(1):123-125.
• [4] GAMBINO G,GRIBAUDO I. Genetic transformation of fruit trees:current status and remaining challenges[J]. Transgenic Research,2012,21(6):1163-1181.
• [5] JIA H F,CHAI Y M,LI C L,QIN L,SHEN Y Y. Cloning and characterization of the H subunit of a magnesium chelatase gene(PpCHLH)in peach[J]. Journal of Plant Growth Regulation,2011,30(4):445-455.
• [6]宋震,李中安,周常勇.病毒诱导的基因沉默(VIGS)研究进展[J].园艺学报,2014,41(9):1885-1894.SONG Zhen,LI Zhong’an,ZHOU Changyong. Research advances of virus-induced gene silencing(VIGS)[J]. Acta Horticulturae Sinica,2014,41(9):1885-1894.
• [7] LI H. BSMV-induced gene silencing assay for functional analysis of wheat rust resistance[J]. Methods in Molecular Biology,2017,1659:257-264.
• [8] MEI Y,ZHANG C,KERNODLE B M,HILL J H,WHITHAM S A. A foxtail mosaic virus vector for virus-induced gene silencing in maize[J]. Plant Physiology,2016,171(2):760-772.
• [9] ULLAH A,SUN H,HAKI M,YANG X,ZHANG X. A novel cotton WRKY gene,GHWRKY6‐like,improves salt tolerance by activating the ABA signaling pathway and scavenging of reactive oxygen species[J]. Physiologia Plantarum,2018,162(4):439-454.
• [10] AGüERO J,VIVES M C,VELáZQUEZ K,PINA J A,NAVARRO L,MORENO P,GUERRI J. Effectiveness of gene silencing induced by viral vectors based on citrus leaf blotch virus is different in nicotiana benthamiana and citrus plants[J]. Virology,2014,460/461:154-164.
• [11] BENKOVICS A H,NYIKóT,MéRAI Z,SILHAVY D,BISZTRAY G D. Functional analysis of the grapevine paralogs of the SMG7 NMD factor using a heterolog VIGS-based gene depletion-complementation system[J]. Plant Molecular Biology,2011,75(3):277-290.
• [12]熊作明,周春华,陶俊.不同类型枇杷果实着色期间果肉类胡萝卜素含量的变化[J].中国农业科学,2007,40(12):2910-2914.XIONG Zuoming,ZHOU Chunhua,TAO Jun. Changes of carotenoid content in pulp of different loquat types during fruit coloring[J]. Scientia Agricultura Sinica,2007,40(12):2910-2914.
• [13]张东,滕元文.红梨资源及其果实着色机制研究进展[J].果树学报,2011,28(3):485-492.ZHANG Dong,TENG Yuanwen. Germplasm resources of red pears and research progress in pear coloring mechanism[J]. Journal of Fruit Science,2011,28(3):485-492.
• [14] JIN W,WANG H,LI M,LI M,WANG J,YANG Y,ZHANG X,YAN G,ZHANG H,LIU J,ZHANG K. The R2R3 MYB transcription factor PavMYB10.1 involves in anthocyanin biosynthesis and determines fruit skin colour in sweet cherry(Prunus avium L.)[J]. Plant Biotechnology Journal,2016,14(11):2120-2133.
• [15]亓希武,帅琴,范丽,曾其伟,向仲怀,何宁佳.桑树花青素合成酶(ANS)基因的克隆及在2种果色桑树中的表达特征[J].蚕业科学,2013,39(1):5-13.QI Xiwu,SHUAI Qin,FAN Li,ZENG Qiwei,XIANG Zhonghuai,HE Ningjia. Molecular cloning and expressional pattern of anthocyanidin synthase gene in two mulberry species with different fruit colors[J]. Science of Sericulture,2013,39(1):5-13.
• [16]姚冉,石美丽,潘沈元,沈桂芳,张志芳.农杆菌介导的植物遗传转化研究进展[J].生物技术进展,2011,1(4):260-265.YAO Ran,SHI Meili,PAN Shenyuan,SHEN Guifang,ZHANG Zhifang. Progress on Agrobacterium tumefaciens-mediated plant transformation[J]. Current Biotechnology,2011,1(4):260-265.
• [17]方锐,畅飞,孙照霖,李宁,孟庆勇. CRISPR/Cas9介导的基因组定点编辑技术[J].生物化学与生物物理进展,2013,40(8):691-702.FANG Rui,CHANG Fei,SUN Zhaolin,LI Ning,MENG Qingyong. New method of genome editing derived from CRISPR/Cas9[J]. Progress in Biochemistry and Biophysics,2013,40(8):691-702.
• [18]季娜娜,闵德栋,邵淑君,李富军,张新华. VIGS载体在蔬菜作物中的应用研究进展[J].植物生理学报,2016,52(6):810-816.JI Nana,MIN Dedong,SHAO Shujun,LI Fujun,ZHANG Xinhua. Progress of research on application of VIGS vectors in vegetables[J]. Plant Physiology Journal,2016,52(6):810-816.
• [19]舒庆艳,朱瑾,门思琦,郝青,王倩玉,刘政安,曾秀丽,王亮生.基于牡丹类黄酮糖基转移酶基因建立VIGS技术体系[J].园艺学报,2018,45(1):168-176.SHU Qingyan,ZHU Jin,MEN Siqi,HAO Qing,WANG Qianyu,LIU Zheng’an,ZENG Xiuli,WANG Liangsheng. Establishing virus induced gene silencing(VIGS)system in tree peony using PsUFGT genes[J]. Acta Horticulturae Sinica,2018,45(1):168-176.
• [20] BURCH-SMITH T,ANDERSON J,MARTIN G,DINESH-KUMAR S. Application and advantages of virus-induced gene silencing for gene function studies in plants[J]. The Plant Journal,2004,39(5):734-746.