|本期目录/Table of Contents|

[1]陈裕坤,程春振,张梓浩,等.一种龙眼快速遗传转化方法的建立[J].应用与环境生物学报,2020,26(06):1540-1545.[doi:DOI: 10.19675/j.cnki.1006-687x.2019.11008]
 CHEN Yukun,CHENG Chunzheng,ZHANG Zihao & LAI Zhongxiong.Establishment of a rapid transgenic method on longan seedling[J].Chinese Journal of Applied & Environmental Biology,2020,26(06):1540-1545.[doi:DOI: 10.19675/j.cnki.1006-687x.2019.11008]
点击复制

一种龙眼快速遗传转化方法的建立()
分享到:

《应用与环境生物学报》[ISSN:1006-687X/CN:51-1482/Q]

卷:
26卷
期数:
2020年06期
页码:
1540-1545
栏目:
研究论文
出版日期:
2020-12-25

文章信息/Info

Title:
Establishment of a rapid transgenic method on longan seedling
作者:
陈裕坤程春振张梓浩赖钟雄
福建农林大学园艺植物生物工程研究所 福州 350002
Author(s):
CHEN Yukun CHENG Chunzheng ZHANG Zihao & LAI Zhongxiong?
Institute of Horticultural Biotechnology, Fujian Agriculture and Forestry University, Fuzhou 350002, China
关键词:
龙眼实生幼苗根癌农杆菌介导遗传转化
Keywords:
Dimocarpus longan soil seedling Agrobacterium tumefaciens mediated genetic transformation
DOI:
DOI: 10.19675/j.cnki.1006-687x.2019.11008
摘要:
针对龙眼遗传转化传统方法存在转化效率低、周期长、转化的胚状体再生困难以及转化植株生长异常等问题,以龙眼土播实生幼苗为受体材料,采用去顶芽(保留真叶)和去顶法(切除带叶的上部)进行创伤,利用根癌农杆菌介导法侵染伤口,通过抗性筛选获得正常生长发育的再生植株,经PCR扩增、目标片段测序及qRT-PCR检测鉴定阳性转化植株. 结果显示,去顶芽法获得的抗性芽GUS基因阳性检测率33.84%、特异片段(35S-mft-intron)的阳性率为20.83%,去顶法获得的抗性芽GUS基因阳性检测率32.5%;经测序验证,PCR扩增产物的序列与载体特异片段的相似度为100%,外源基因已经整合到龙眼基因组中;qRT-PCR表明外源基因在转基因龙眼植株中表达量较高,在未转化龙眼植株中不表达,说明外源基因在龙眼植株内进行转录,建立了一种龙眼快速转基因方法. 该方法可快速获得大量转基因植株,具有操作简便、成本低、周期短、转化效率较高、成活率高、可周年生产等优点,对龙眼的基因功能研究和种质创新具有重要的意义. (图4 参28)
Abstract:
Traditional longan genetic transformation methods have a series of problems, such as low transformation efficiency, long cycles, transformed embryoids that are difficult to regenerate, and abnormal growth of transformed plants. In this study, we transformed the target genes into longan soil seedlings by Agrobacterium tumefaciens-mediated transformation through infection of a wound, generated by removing the apical bud (keep true leaf) or the top (remove the upper part of the true leaf) of the seedling. Many normal, resistant buds were obtained through hygromycin selection, and successfully transformed plants were identified by polymerase chain reaction (PCR), sequencing, and quantitative reverse-transcription PCR (qRT-PCR). PCR detection of target genes showed that the successful transformation rate of GUS in resistant shoots was 33.84% and 32.5% following removal of the apical bud and the top of the seedling, respectively. The successful transformation rate of a specific fragment (35S-mft-intron) in resistant shoots was 20.83% by removing the apical bud of the seedling. It was verified by sequencing that the similarity between the PCR amplification product and the specific fragment of the vector was 100%, indicating that the target genes were integrated into longan gDNA. qRT-PCR analysis suggested that exogenous genes were highly expressed in transgenic longan plants but not in the negative control, which confirmed that the target transformation genes in longan were being transcribed. This method can produce a large number of transgenic longan trees rapidly, and it may play a significant role in gene functional studies and the creation of longan germplasm, owing to its easy, rapid, and efficient production, as well as the high survival rate of transgenic plants.

参考文献/References:

1 赖钟雄, 陈振光. 龙眼胚性愈伤组织的高频率体细胞胚胎发生[J]. 福建农林大学学报(自然科学版), 1997, 26 (3): 271-276 [Lai ZX, Chen ZG. Somatic embryogenesis of high frequency from longan embryogenic calli [J]. J FAFU (Nat Sci Ed), 1997, 26 (3): 271-276]
2 赖钟雄, 潘良镇, 陈振光. 龙眼胚性细胞系的建立与保持[J]. 福建农林大学学报(自然科学版), 1997, 26 (2): 160-167 [Lai ZX, Pan LZ, Chen ZG. Establishment and maintenance of longan embryogenic cell lines [J]. J FAFU (Nat Sci Ed), 1997, 26 (2): 160-167]
3 赖钟雄, 陈振光. 龙眼胚性细胞悬浮培养再生植株[J]. 应用与环境生物学报, 2002, 8 (5): 485-491 [Lai ZX, Chen ZG. Plant regeneration from embryogenic cell suspensions via somatic embryogenesis of longan (Dimocarpus longan Lour.) [J]. Chin J Appl Environ Biol, 2002, 8 (5): 485-491]
4 赖钟雄, 陈振光. 龙眼胚性悬浮细胞原生质体培养及其体胚发生再生植株[J]. 热带作物学报, 2002, 23 (3): 53-60 [Lai ZX, Chen ZG. Plant regeneration from protoplasts isolated from embryogenic suspension cells via somatic embryogenesis in longan [J]. Chin J Trop Crops, 2002, 23 (3): 53-60]
5 赖钟雄, 陈振光. 龙眼单细胞培养及其体胚发生再生植株[J]. 热带作物学报, 2003, 24 (2): 16-18 [Lai ZX, Chen ZG. Plant regeneration from in vitro culture of single cell via somatic embryogenesis in longan (Dimocarpus longan Lour.) [J]. Chin J Trop Crops, 2003, 24 (2): 16-18]
6 曾黎辉. 龙眼遗传转化研究[D]. 福州: 福建农林大学, 1998 [Zeng LH. Study on genetic transformation of longan [D]. Fuzhou: Fujian Agriculture and Forestry University, Fujian Agriculture University, 1998]
7 曾黎辉, 陈振光, 吕柳新. 发根农杆菌转化龙眼研究初报[J]. 福建农业大学学报, 2000, 29 (1): 27-30 [Zeng LH, Chen ZG, Lü LX. A preliminary report on Agrobacterium rhizogenes mediated transformation of longan [J]. J FAFU, 2000, 29 (1): 27-30]
8 Zeng LH, Chen ZG, Lu LX, Zhou P, Zheng XQ. In vitro transformation mediated by Ri plasmid of Agrobacterium rhizogenes and transgenic plant regeneration of longan [J]. Acta Hortic. 558, 2001: 149-155
9 赖钟雄. 龙眼生物技术研究[M]. 福州: 福建科学技术出版社, 2003: 53-54 [Lai ZX. Study on the biotechnology of longan [M]. Fuzhou: Fujian Science and Technology Publishing House, 2003: 53-54]
10 张妙霞. 香蕉与龙眼转化受体系统建立及转化PEAS基因初步研究[D]. 福州: 福建农林大学, 2004 [Zhang MX. Establishment of banana and longan transgenic receptor system and the preliminary study on the transformation of the PEAS gene [D]. Fuzhou: Fujian Agriculture and Forestry University, 2004]
11 Zheng QF, Hu GB, Chen DC, Huang ZR. A study of cell suspension culture and genetic transformation of litchi and longan [J]. J Fruit Sci, 2005, 22 (2): 125-128.
12 徐清锋. 龙眼体胚发生系统的优化及其在种质保存与遗传转化上的应用[D]. 福州: 福建农林大学, 2010 [Xu QF. The optimization of somatic embryogenesis system and its applications to germplasm conservation and genetic transformation in Dimocarpus longan Lour. [D]. Fuzhou: Fujian Agriculture and Forestry University, 2010]
13 曾丽兰. 龙眼胚性愈伤组织SOD的表达分析及启动子功能鉴定[D]. 福州: 福建农林大学, 2013 [Zeng LL. Characterization of SOD promoters and expression analysis of SOD genes of the embryogenic callus in Dimocarpus longan Lour. [D]. Fuzhou: Fujian Agriculture and Forestry University, 2013]
14 Tepfer D. Transformation of several species of higher plants by agrobacterium rhizogenes: sexual transmission of the transformed genotype and phenotype [J]. Cell, 1984, 37 (3): 959-967
15 Feldmann KA, Marks MD. Agrobacterium-mediated transformation of germinating seeds of Arabidopsis thaliana: a non-tissue culture approach [J]. Mol Genet Genomics, 1987, 208: 1-9
16 Clough SJ, Bent AF. Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana [J]. Plant J, 1998, 16 (6): 735-743
17 Jones HD, Doherty A, Wu H. Review of methodologies and a protocol for the Agrobacterium-mediated transformation of wheat [J]. Plant methods, 2005, 1: 5
18 Janice M Zale, Agarwal S, Loar S. Evidence for stable transformation of wheat by floral dip in Agrobacterium tumefaciens [J]. Plant Cell Rep, 2009, 28: 903-913
19 Jaganath B, Subramanyam K, Mayavan S, Karthik S, Elayaraja D, Udayakumar R, Manickavasagam M, Ganapathi A. An efficient in planta transformation of Jatropha curcas (L.) and multiplication of transformed plants through in vivo grafting [J]. Protoplasma, 2014, 251 (3): 591-601
20 Shah SH, Ali S, Jan SA, Jalal-Ud-Din, Ali GM. Piercing and incubation method of in planta transformation producing stable transgenic plants by overexpressing DREB1A gene in tomato (Solanum lycopersicum Mill.) [J]. Plant Cell Tiss Org, 2015, 120 (3): 1139-1157
21 Zhong LH, Zhang YP, Liu HC, Sun GW, Chen RY, Song SW. Agrobacterium-mediated transient expression via root absorption in flowering Chinese cabbage [J]. Springer Plus, 2016, 5 (1): 1825
22 Chen L, Cai YP, Liu XJ, Yao WW, Guo C, Sun S, Wu CX, Jiang BJ, Han TF, Hou WS. Improvement of Soybean Agrobacterium-mediated transformation efficiency by adding glutamine and asparagine into the culture media [J]. Int J Mol Sci, 2018, 19 (10): 3039
23 Zhang YY, Zhang DM, Zhong Y, Chang XJ, Hu ML, Cheng CZ. A simple and efficient in planta transformation method for pommelo (Citrus maxima) using Agrobacterium tumefaciens [J]. Sci Hortic-Amsterdam, 2017, 214: 174-179
24 谢幸男, 杨莉, 刘范, 田娜, 车婧如, 靳三鹏, 张永艳, 程春振.‘伏令夏橙’原位转化体系的建立及优化[J]. 园艺学报. doi: 10.16420/j.issn.0513-353x.2019-0130 [Xie XN, Yang L, Liu F, Tian N, Che JR, Jin SP, Zhang YY, Cheng CZ. Establishment and optimization of Valencia sweet orange in planta transformation system [J]. Acta Hortic Sin, 2019, doi: 10.16420/j.issn.0513-353x]
25 Chen YK, Xu XH, Chen XH, Chen Y, Zhang ZH, Xu XH, Lin YL, Lai ZX. Seed-specific gene mother of FT and TFL1 (MFT) involved in embryogenesis, hormones and stress responses in Dimocarpus longan Lour. [J]. Int J Mol Sci, 2018, 19 (8): 2403
26 Lin YL, Lai ZX. Reference gene selection for qPCR analysis during somatic embryogenesis in longan tree [J]. Plant Sci, 2010, 178 (4): 359-365
27 陈春玲, 赖钟雄. 龙眼胚性愈伤组织体胚发生同步化调控及组织细胞学观察[J]. 福建农林大学学报(自然科学版), 2002, 31 (2): 192-194 [Chen CL, Lai ZX. Synchronization regulation of embryogenesis of embryogenic calli and their histological observations in longan [J]. J FAFU (Nat Sci Ed), 2002, 31 (2): 192-194]
28 田奇琳. 龙眼胚性培养物DlRan3A和DlRan3B基因的功能分析[D]. 福州: 福建农林大学, 2017 [Tian QL. Characterization of DlRan3A and DlRan3B gene from embryogenic cultures in Dimocarpus longan Lour. [D]. Fuzhou: Fujian Agriculture and Forestry University, 2017]

相似文献/References:

[1]邱栋梁,刘星辉,王湘平.模拟酸雨对龙眼叶绿体的伤害效应[J].应用与环境生物学报,2002,8(02):154.
 QIU Dongliang,et al..Injury effects of simulated acid rain on chloroplasts of longan leaves[J].Chinese Journal of Applied & Environmental Biology,2002,8(06):154.
[2]邱栋梁,刘星辉,郭素枝.模拟酸雨对龙眼幼果纤维素酶活性和内源激素含量的影响[J].应用与环境生物学报,2004,10(01):35.
 QIU Dongliang,et al..Effects of simulated acid rain on cellulase activity and contents of endogenous hormone in young fruit of longan[J].Chinese Journal of Applied & Environmental Biology,2004,10(06):35.
[3]林玉玲,赖钟雄.龙眼胚性愈伤组织Cu/Zn-SOD分子伴侣基因CCS的克隆及其在体胚发生过程中的表达分析[J].应用与环境生物学报,2012,18(03):351.[doi:10.3724/SP.J.1145.2012.00351]
 LIN Yuling,LAI Zhongxiong.Cloning of Copper Chaperone for Superoxide Dismutase Gene CCS from Embryogenic Callus of Dimocarpus longan Lour. and Its Expression Analysis During Somatic Embryogenesis[J].Chinese Journal of Applied & Environmental Biology,2012,18(06):351.[doi:10.3724/SP.J.1145.2012.00351]
[4]赖瑞联,林玉玲,赖钟雄.龙眼生长素受体基因TIR1的克隆及其与miR393互作关系[J].应用与环境生物学报,2016,22(01):95.[doi:10.3724/SP.J.1145.2015.05051]
 LAI Ruilian,LIN Yuling & LAI Zhongxiong**.Cloning of auxin receptor gene TIR1 and its interaction with miR393 in Dimocarpus longan Lour.[J].Chinese Journal of Applied & Environmental Biology,2016,22(06):95.[doi:10.3724/SP.J.1145.2015.05051]
[5]陈旭,曾友竞,王嘉毅,等.龙眼miR159家族成员进化特性及时空表达[J].应用与环境生物学报,2017,23(04):602.[doi:10.3724/SP.J.1145.2017.03011]
 CHEN Xu,ZENG Youjing,WANG Jiayi,et al.Effect of main grain components on the starch swelling power of Tibetan hull-less barley (Hordeum vulgare var. nudum)[J].Chinese Journal of Applied & Environmental Biology,2017,23(06):602.[doi:10.3724/SP.J.1145.2017.03011]
[6]王云,彭丽云,孙雪丽,等.龙眼Hsf基因家族全基因组鉴定及体胚发生过程中的表达分析[J].应用与环境生物学报,2019,25(02):420.[doi:10.19675/j.cnki.1006-687x.2018.06004]
 WANG Yun,PENG Liyun,SUN Xueli,et al.Genome-wide identification of longan Hsf family members and their functional analysis during somatic embryogenesis in longan[J].Chinese Journal of Applied & Environmental Biology,2019,25(06):420.[doi:10.19675/j.cnki.1006-687x.2018.06004]
[7]苏立遥,黄倏祺,蒋梦琦,等.龙眼miR403及其候选靶标对外源激素的响应模式以及在龙眼体胚中的表达模式[J].应用与环境生物学报,2019,25(04):977.[doi:10.19675/j.cnki.1006-687x.2019.03058]
 SU Liyao,HUANG Shuqi,JIANG Mengqi,et al.The response patterns of miR403 and its candidate targets to exogenous hormones and their expression profiles in the longan somatic embryo[J].Chinese Journal of Applied & Environmental Biology,2019,25(06):977.[doi:10.19675/j.cnki.1006-687x.2019.03058]
[8]李汉生,孙刚,陈晓慧,等.龙眼BRI1基因家族的全基因组鉴定及光照响应表达[J].应用与环境生物学报,2020,26(01):125.[doi:10.19675/j.cnki.1006-687x.2019.07053]
 LI Hansheng,SUN Gang,CHEN Xiaohui,et al.Genome-wide identification and response light expression analysis of the BRI1 family in Dimocarpus longan Lour[J].Chinese Journal of Applied & Environmental Biology,2020,26(06):125.[doi:10.19675/j.cnki.1006-687x.2019.07053]
[9]刘蒲东,张舒婷,陈晓慧,等.龙眼GRF家族全基因组鉴定及表达模式[J].应用与环境生物学报,2020,26(02):236.[doi:10.19675/j.cnki.1006-687x.2019.06028]
 LIU Pudong,ZHANG Shuting,CHEN Xiaohui,et al.Genomic identification and expression patterns of the longan GRF family[J].Chinese Journal of Applied & Environmental Biology,2020,26(06):236.[doi:10.19675/j.cnki.1006-687x.2019.06028]
[10]廖斌,徐小萍,李珊珊,等.苯丙氨酸和茉莉酸甲酯对龙眼胚性悬浮细胞柯里拉京积累的影响[J].应用与环境生物学报,2020,26(02):287.[doi:10.19675/j.cnki.1006-687x.2019.06001]
 LIAO Bin,XU Xiaoping,LI Shanshan,et al.Effects of phenylalanine and methyl jasmonate on the growth and corilagin accumulation of embryogenic suspension cells in Dimocarpus longan Lour.[J].Chinese Journal of Applied & Environmental Biology,2020,26(06):287.[doi:10.19675/j.cnki.1006-687x.2019.06001]

更新日期/Last Update: 2020-12-25