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[1]孙家旗,唐维,刘永胜. 猕猴桃CHS基因RNA干涉载体在果实中的瞬时表达可以有效影响花青素积累[J].应用与环境生物学报,2014,20(05):929-933.[doi:10.3724/SP.J.1145.2014.03021]
 SUN Jiaqi,TANG Wei,LIU Yongsheng. Transient expression of CHS-RNAi effectively influences the accumulation of anthocyanin in fruit of kiwifruit (Actinidia chinensis)[J].Chinese Journal of Applied & Environmental Biology,2014,20(05):929-933.[doi:10.3724/SP.J.1145.2014.03021]
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 猕猴桃CHS基因RNA干涉载体在果实中的瞬时表达可以有效影响花青素积累()
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《应用与环境生物学报》[ISSN:1006-687X/CN:51-1482/Q]

卷:
20卷
期数:
2014年05期
页码:
929-933
栏目:
研究简报
出版日期:
2014-10-25

文章信息/Info

Title:
 Transient expression of CHS-RNAi effectively influences the accumulation of anthocyanin in fruit of kiwifruit (Actinidia chinensis)
作者:
 孙家旗唐维刘永胜
 猕猴桃;CHS;花青素;瞬时表达;RNAi;qRT-PCR
Author(s):
 SUN Jiaqi TANG Wei LIU Yongsheng
1Ministry of Education Key Laboratory for Bio-resource and Eco-environment, College of Life Sciences, State Key Laboratory of Hydraulics and Mountain River Engineering, Sichuan University, Chengdu 610064, China,
2School of Biotechnology and Food Engineering, Hefei University of Technology, Hefei 230009, China
关键词:
1四川大学生命科学学院生物资源与生态环境教育部重点实验室水力学与山区河流工程国家重点实验室 成都 6100642合肥工业大学生物技术与食品工程学院 合肥 230009
Keywords:
 kiwifruit CHS anthocyanin transient expression qPCR
分类号:
S663.403
DOI:
10.3724/SP.J.1145.2014.03021
文献标志码:
A
摘要:
近来猕猴桃基因组草图已经完成,然而对猕猴桃功能基因的研究还非常有限. 我们拟建立RNA干扰(RNAi)系统来研究猕猴桃的基因功能. 将红阳猕猴桃中编码查耳酮合成酶的CHS基因片段插入到载体pHB 和pTCK303中,构建35S::CHS-RNAi 和UBI::CHS-RNAi载体. 将两个重组质粒转入根癌土壤杆菌EHA105中,分别将0.7 mL农杆菌(D = 0.8)从果实底部注入花后106 d(果肉颜色开始变红)的猕猴桃果实中. 5 d后,与空载体相比,注射了CHS-RNAi载体的猕猴桃果肉颜色变浅,花青素含量、CHS的表达量显著降低. 预测这种快捷的瞬时表达系统将为猕猴桃基因功能研究提供有效手段.
Abstract:
Functional genomic studies on kiwifruit are very limited despite the fact that its genomic data is available. In the present study, we intended to establish an efficient system to investigate gene function by using RNA interference (RNAi) technique. A segment of cDNA putatively coding for chalcone synthase (CHS) derived from kiwifruit cv. Hongyang (Actinidia chinensis) was inserted into vectors pHB and pTCK303, to produce recombinant constructs 35S::CHS-RNAi and UBI::CHS-RNAi, respectively. The two recombinant plasmids were transformed into Agrobacterium tumefaciens EHA105 by liquid nitrogen. These two engineered A. tumefaciens strains were subsequently injected into the kiwifruits 106 days after full bloom (DAFB) when the red pigment of inner pericarp started to accumulate. About 0.7 mL engineered A. tumefaciens culture (about OD 0.8) was injected through fruit bottom into core tissue. As a result, 5 days after injection, as compared with blank control, a significant reduction of red pigment accumulation was visualized in inner pericarps injected with the engineered A. tumefaciens strains containing CHS-RNAi vectors, with remarkably decreased contents of anthocyanins detected at the same time. In addition, the CHS expression level was lower with the RNAi repression than that injected with empty vector. We anticipate that this rapid and transient expression system will be useful for studying the function of plant structural and regulatory genes in kiwifruit.

参考文献/References:

1 Hunter CP. Gene silencing: shrinking the black box of RNAi [J]. Curr Biol, 2000, 10 (4): 137-140<br/>

2 Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells [J]. Nature, 2001, 411 (6836): 494-498<br/>

3 Bass BL. RNA interference: the short answer [J]. Nature, 2001, 411 (6836): 428-429<br/>

4 Harborth J, Elbashir SM, Bechert K,Tuschl T, Weber K. Identification of essential genes in cultured mammalian cells using small interfering RNAs [J]. J Cell Sci, 2001, 114 (24): 4557-4565<br/>

5 刘继恺, 高永峰, 牛向丽, 刘永胜. 番茄HP1HP2基因RNA共干涉载体的构建及遗传转化[J]. 应用与环境生物学报, 2009, 15 (5): 591-595 [Liu JK, Gao YF, Niu XL, Liu YS. Construction and transformation of Co-RNAi vector of tomato HP1 and HP2 genes [J]. Chin J Appl Environ Biol, 2009, 15 (5): 591-595]<br/>

6 Wang S, Liu J, Feng Y, Niu X, Giovannoni J, Liu Y. Altered plastid levels and potential for improved fruit nutrient content by downregulation of the tomato DDB1-interacting protein CUL4 [J]. Plant J, 2008, 55 (1): 89-103<br/>

7 Niu X, Tang W, Huang W, Ren G, Wang Q, Luo D. RNAi-directed downregulation of OsBADH2 results in aroma (2-acetyl-1-pyrroline) production in rice (Oryza sativa L.) [J]. BMC Plant Biol, 2008, 8 (1): 92-100<br/>

8 邱礽, 陶刚, 李奇科, 邱又彬, 刘作易. 农杆菌渗入法介导的基因瞬时表达技术及应用[J]. 分子植物育种, 2009, 7 (5): 1032-1039 [Qiu R, Tao G, Li QK, Liu ZY. Transient gene expression mediated by agroinfiltration and its application [J]. Mol Plant Breed, 2009: 7 (5): 1032-1039]<br/>

9 Kapila J, De Rycke R, Van Montagu M, Angenon G. An Agrobacterium-mediated transient gene expression system for intact leaves [J]. Plant Sci, 1997, 122 (1): 101-108<br/>

10 Bulley SM, Rassam M, Hoser D,Otto W, Schünemann N, Wright M, Macrae E, Gleave A, Laing, W. Gene expression studies in kiwifruit and gene overexpression in Arabidopsis indicates that GDP-L-galactose guanyltransferase is a major control point of vitamin C biosynthesis [J]. J Exp Bot, 2009, 60 (3): 765-778<br/>

11 Chen Z, Young TE, Ling J,Chang SC, Gallie DR. Increasing vitamin C content of plants through enhanced ascorbate recycling [J]. PNAS, 2003, 100 (6): 3525-3530<br/>

12 Choi JJ, Klosterman SJ, Hadwiger LA. A promoter from pea gene DRR206 is suitable to regulate an elicitor-coding gene and develop disease resistance [J]. Phytopathology, 2004, 94 (6): 651-660<br/>

13 Hoffmann T, Kalinowski G, Schwab W. RNAi-induced silencing of gene expression in strawberry fruit (Fragaria × ananassa) by agroinfiltration: a rapid assay for gene function analysis [J]. Plant J, 2006, 48 (5): 818-826<br/>

14 Santos-Rosa M, Poutaraud A, Merdinoglu D, Mestre P. Development of a transient expression system in grapevine via agroinfiltration [J]. Plant Cell Rep, 2008, 27 (6): 1053-1063<br/>

15 Sohi HH, Jourabchi E, Khodabandeh M. Transient expression of human growth hormone in potato (Solanum tuberosum), tobacco (Nicotiana tobacum) and lettuce (Lactuca sativa) leaves by agroinfiltration [J]. Iranian J Biotechnol, 2005, 3 (2): 109<br/>

16 Voinnet O, Pinto YM, Baulcombe DC. Suppression of gene silencing: a general strategy used by diverse DNA and RNA viruses of plants [J]. PNAS, 1999, 96 (24): 14147-14152<br/>

17 Yang Y, Li R, Qi M. In vivo analysis of plant promoters and transcription factors by agroinfiltration of tobacco leaves [J]. Plant J, 2000, 22 (6): 543-551<br/>

18 Montefiori M, Espley RV, Stevenson D, Cooney J, Datson PM, Saiz A. Identification and characterisation of F3GT1 and F3GGT1, two glycosyltransferases responsible for anthocyanin biosynthesis in red-fleshed kiwifruit (Actinidia chinensis) [J]. Plant J, 2011, 65 (1): 106-118<br/>

19 Griesser M, Hoffmann T, Bellido ML, Rosati C, Fink B, Kurtzer R. Redirection of flavonoid biosynthesis through the down-regulation of an anthocyanidin glucosyltransferase in ripening strawberry fruit [J]. Plant Physiol, 2008, 146 (4): 1528-1539<br/>

20 Miyawaki K, Fukuoka S, Kadomura Y, Hamaoka H, Mito T, Ohuchi H. Establishment of a novel system to elucidate the mechanisms underlying light-induced ripening of strawberry fruit with an Agrobacterium-mediated RNAi technique [J]. Plant Biotechnol, 2012, 29 (3): 271-277<br/>

21 Jaakola L, Poole M, Jones M O. A SQUAMOSA MADS box gene involved in the regulation of anthocyanin accumulation in bilberry fruits [J]. Plant Physiol, 2010, 153 (4): 1619-1629<br/>

22 Jiang F, Wang JY, Jia HF, Jia WS, Wang HQ, Xiao M. RNAi-mediated silencing of the flavanone 3-hydroxylase gene and its effect on flavonoid biosynthesis in strawberry fruit [J]. Plant Grow Regul, 2013, 32 (1): 182-190<br/>

23 Xie XB, Li S, Zhang RF, Zhao J, Chen YC, Zhao Q. The bHLH transcription factor MdbHLH3 promotes anthocyanin accumulation and fruit colouration in response to low temperature in apples [J]. Plant Cell Environ, 2012, 35 (11): 1884-1897<br/>

24 Hanania U, Velcheva M, Or E, Flaishman M, Sahar N, Perl A. Silencing of chaperonin 21, that was differentially expressed in inflorescence of seedless and seeded grapes, promoted seed abortion in tobacco and tomato fruits [J]. Transgenic Res, 2007, 16 (4): 515-525<br/>

25 Huang S, Ding J, Deng D, Tang W, Sun H, Liu Y. Draft genome of the kiwifruit Actinidia chinensis [J]. Nature Commun, 2013, 10 (4): 2640<br/>

26 Wang S, Jiang Z, Zhang Z, Gong J, Huang H. Exploration of Actinidia genetic resources and development of kiwifruit industry in China [J]. Intern Symp Kiwifruit, 2002, 610: 29-43<br/>

27 Nishiyama I, Yamashita Y, Yamanaka M, Shimohashi A, Fukuda T, Oota T. Varietal difference in vitamin C content in the fruit of kiwifruit and other Actinidia species [J]. J Agric Food Chem, 2004, 52 (17): 547-5475<br/>

28 Montefiori M, McGhie TK, Costa G, Ferguson AR. Pigments in the fruit of red-fleshed kiwifruit (Actinidia chinensis and Actinidia deliciosa) [J]. J Agric Food Chem, 2005, 53 (24): 9526-9530<br/>

29 Martin CR. Structure, function, and regulation of the chalcone synthase [J]. Inter Rev Cytol, 1993, 147: 233-284<br/>

30 Koes RE, Quattrocchio F, Mol JNM. The flavonoid biosynthetic pathway in plants: function and evolution [J]. BioEssays, 1994, 16 (2): 123-132<br/>

31 Wang Z, Chen C, Xu Y, Jiang R, Han Y, Xu Z, Chong K. A practical vector for efficient knockdown of gene expression in rice (Oryza sativa L.) [J]. Plant Mol Biol Rep, 2004, 22 (4): 409-417<br/>

32 Yu H, Yin J, Li H, Yang S, Shen Z. Construction and selection of the novel recombinant Escherichia coli strain for poly (β-hydroxybutyrate) production [J]. J Biosci Bioeng, 2000, 89 (4): 307-311<br/>

33 孟楠, 刘月学, 孙立茹, 杜安楠, 李贺, 张志宏. 农杆菌注射法转化草莓果实细胞的研究[J]. 沈阳农业大学学报, 2009, 40 (4): 404-407 [Meng N, Liu YX, Sun LN, Du AN, Li H, Zhang ZH. Transformation of cells in strawberry fruit with injection of Agrobacterium [J]. J.Shenyang Agric Univ, 2009, 40 (4): 404-407]<br/>

34 Fuleki T, Francis FJ. Quantitative methods for anthocyanins [J]. J Food Sci, 1968, 33 (3): 266-274<br/>

35 Li M, Ma F, Liang D, Li J, Wang Y. Ascorbate biosynthesis during early fruit development is the main reason for its accumulation in kiwi [J]. PloS ONE, 2010, 5 (12): e14281

备注/Memo

备注/Memo:
 四川省科技厅科技支撑项目(2013NZ0014)和973计划重大项目(2011CB100401)资助 Supported by the Key Project from the Government of Sichuan Province (2013NZ0014), and the National Basic Research Program of China (973 Program) (2011CB100401)
更新日期/Last Update: 2014-10-30