不同甜瓜品种遗传转化再生体系的建立与基因编辑植株的快速获取

doi:10.6048/j.issn.1001-4330.2024.07.013

新疆农业科学 ›› 2024, Vol. 61 ›› Issue (7): 1666-1672.DOI: 10.6048/j.issn.1001-4330.2024.07.013

• 园艺特产·贮藏保鲜加工 • 上一篇下一篇

不同甜瓜品种遗传转化再生体系的建立与基因编辑植株的快速获取

卡地尔阿依·买买提¹(), 周婷婷¹, 韩盛¹, 梅丽克汗·热西提², 玉山江·麦麦提¹()

1.新疆农业科学院植物保护研究所/农业农村部西北荒漠绿洲作物有害生物综合治理重点实验室,乌鲁木齐 830091
2.哈密市农业农机技术推广服务中心,新疆哈密 839000

收稿日期:2023-11-15 出版日期:2024-07-20 发布日期:2024-09-04
通信作者: 玉山江·麦麦提(1978-),男,新疆人,研究员,博士,硕士生导师,研究方向为瓜类作物病虫害防治,(E-mail)yusanjan418@163.com
作者简介:卡地尔阿依·买买提(1996-),女,新疆人,硕士研究生,研究方向为植物病虫害防治,(E-mail)2432443698@qq.com
基金资助:
国家自然科学基金地区科学基金项目“基于CRISPR/Cas9技术的哈密瓜抗多病毒病非转基因材料创制研究”(31760511)

Establishment of genetic transformation and regeneration systems for different melon varieties and rapid acquisition of gene edited plants

Kadierayi Maimaiti¹(), ZHOU Tingting¹, HAN Sheng¹, Meilikehan Rexiti², Yushanjiang Maimaiti¹()

1. Key Laboratory of Integrated Pest Management on Crops in Northwestern Oasis,Ministry of Agriculture and Rural Affairs, P. R. China / Institute of Plant Protection,Xinjiang Academy of Agricultural Sciences,Urumqi 830091, China
2. Hami Agricultural Machinery Technology Promotion Service Center, Hami Xinjiang 839000,China

Received:2023-11-15 Published:2024-07-20 Online:2024-09-04
Supported by:
The Regional Science Foundation project of the National Natural Science Foundation of China "Study on Polyvirus Disease Based on Crispr / Cas 9 Technology"(31760511)

摘要/Abstract

摘要：

【目的】研究不同甜瓜品种遗传转化再生体系的建立与基因编辑植株的快速获取。【方法】选用巴吾顿、老汉瓜、其里甘、新密杂11号(86-1)4个甜瓜品种的子叶为外植体,通过农杆菌介导法侵染甜瓜子叶,采用组织培养法建立老汉瓜的遗传转化再生体系,运用PCR法检测获取基因编辑植株。【结果】从4个甜瓜品种中筛选出老汉瓜为优势品种作为外植体,在MS+6-BA 1.0 mg/L+NAA 0.05 mg/L培养基预培养2 d和共培养3 d,在MS+6-BA 1.0 mg/L+NAA 0.05 mg/L+潮霉素5 mg/L+头孢霉素250 mg/L筛选培养基上继续培养7 d,在MS+6-BA 1.0 mg/L+NAA 0.05 mg/L+潮霉素5 mg/L+头孢霉素250 mg/L的不定芽诱导培养基上培养1~2周可见长出的小芽,将诱导芽分别放置于不加潮霉素和加潮霉素的芽伸长培养基上培养,诱导芽在不加潮霉素的芽伸长培养基上的生长速度比加潮霉素的培养基生长快,可使芽生长至2叶苗期缩短3周,在无菌环境下剪去一个芽提取DNA,并采用RT-PCR方法检测获得21株转化植株。【结论】筛选出基因编辑植株嫩芽,不加潮霉素的芽伸长培养基可将获得基因编辑植株周期缩短3周。

关键词: 甜瓜; 农杆菌介导; 遗传转化; 基因编辑植株

Abstract:

【Objective】Study on establishment of genetic transformation and regeneration systems for different melon varieties and rapid acquisition of gene edited plants.【Methods】The cotyledons of four melon varieties, including Bawudun, Laohangua, Qiligan, and Xinmiza No. 11 (86-1).The sweet melon cotyledons were infected by Agrobacterium tumefaciens mediated method, and a genetic transformation and regeneration system of Laohan melon was established by tissue culture method and then genetically edited plants were obtained through PCR detection.【Results】The results showed that Laohan melon was selected as the dominant variety from four melon varieties as the explants. It was pre-cultured on MS+6-BA 1.0 mg/L+NAA 0.05 mg/L medium for 2 days and co-cultured for 3 days, then it was further cultured on MS+6-BA 1.0 mg/L+NAA 0.05 mg/L+hygromycin 5 mg/L+cephalosporin 250 mg/L screened medium for 7 days, after that, small buds were seen growing on MS+6-BA 1.0 mg/L+NAA 0.05 mg/L+hygromycin 5 mg/L+cephalosporin 250 mg/L adventitious bud induction medium for 1-2 weeks. The induced buds were placed on both non hygromycin and hygromycin added bud elongation medium for cultivation. It was found that the growth rate of the induced buds on the non hygromycin added bud elongation medium was faster than that on the hygromycin added medium, which shortened the growth of the buds to the two leaf seedling stage by 3 weeks. One bud DNA was cut off in a sterile environment and 21 transformed plants were examined by RT-PCR.【Conclusion】Gene edited plant buds were successfully selected on a shoot elongation medium with added hygromycin. The use of a shoot elongation medium without added hygromycin can shorten the time for obtaining gene edited plants by 3 weeks.

Key words: muskmelon; Agrobacterium mediated; genetic transformation; genome edited plant

中图分类号:

S652

卡地尔阿依·买买提, 周婷婷, 韩盛, 梅丽克汗·热西提, 玉山江·麦麦提. 不同甜瓜品种遗传转化再生体系的建立与基因编辑植株的快速获取[J]. 新疆农业科学, 2024, 61(7): 1666-1672.

Kadierayi Maimaiti, ZHOU Tingting, HAN Sheng, Meilikehan Rexiti, Yushanjiang Maimaiti. Establishment of genetic transformation and regeneration systems for different melon varieties and rapid acquisition of gene edited plants[J]. Xinjiang Agricultural Sciences, 2024, 61(7): 1666-1672.

图/表 9

表1 用于抗病性检测的特异性引物

Tab.1 Specific primers for disease resistance detection

引物名称 Primers	引物序列(5'-3') Sequence (5'-3')	扩增片段大小 Product size (bp)
HPT-1F	GAAAAGTTCGACAGCGTCTCC	407
HPT-1R	CGTCCATCACAGTTTGCCAGT	407
HPT-2F	TCGGACGATTGCGTCGCATC	720
HPT-2R	AGGCTATGGATGCGATCGCTG	720
HPT-3F	TGAACTCACCGCGACGTCTGT	980
HPT-3R	TGCGCCCAAGCTGCATCAT	980

表2 PCR反应体系

Tab.2 Primer sequence

组分 Component	体积 Volume(μl)
Mix	10
正向引物 Forward primer	1
反向引物 Reverse primer	1
ddH₂O	7
模板 Template	1
总计Total	20

表3 侵染不同品种出愈率和不定芽诱导上的差异

Tab.3 The difference of recovery rate and adventitious bud induction among infected varieties

品种名称 Variety name	总外植体数(块) Total number of explants (pieces)	出愈数(个) Number of healing points	出愈率 Healing rate (%)	不定芽诱导数(个) Adventitious bud inducing derivative (number)	不定芽诱导率 Adventitious bud induction rate (%)
巴吾顿 Bawudun	420	294	70	9	2.14
老汉瓜 Laohangua	420	362	86.19	36	8.57
其里甘 Qiligan	420	353	84.04	0	0
新密杂11号 Xinmiza 11	420	294	70	17	4.04

表4 侵染不同品种褐化率和不定芽伸长的差异

Tab.4 Differences in Browning rate and adventitive bud elongation among infected varieties

品种名称 Variety name	总外植体数(块) Total number of explants (pieces)	褐化数(个) Browning number (pieces)	褐化率 Browning rate (%)	芽伸长数(个) Number of bud elongation (pieces)	芽伸长率 Bud elongation rate (%)
巴吾顿 Bawudun	420	243	57.85	1	0.23
老汉瓜 Laohangua	420	200	47.61	8	1.90
其里甘Qiligan	420	260	61.90	0	0
新密杂11号 Xinmiza 11	420	220	52.38	1	0.23

图1 基因编辑植株在加潮霉素和未加潮霉素的芽伸长培养基上的生长差异注:A:加潮霉素培养基培养前;B:加潮霉素培养基上培养14 d后;C:未加潮霉素的培养基培养前;D:未加潮霉素的培养基培养14 d后

Fig.1 Growth difference of gene-edited plants on bud elongation medium with and without hygromycin Note: A: Before culturing with hygromycin medium; B: After 2 weeks of cultivation on hygromycin medium; C: Before culturing in a culture medium without adding hygromycin; D: After 2 weeks of cultivation in medium without added hygromycin

图2 基因编辑出芽的基因组提取DNA 注:M:D2000的DNA marker;1~21:是基因编辑植株叶片的DNA

Fig.2 Genome of bud extracted by gene editing Note: M: DNA marker of D2000; 1-21: DNA of leaves of genome editing plants

图3 部分基因编辑植株

Fig.3 Partial gene-edited plants

图4 基因编辑苗的PCR扩增结果注:M:D2000的DNA marker;1~9是SG10592获取的基因编辑苗;10~21是SG10610获取的基因编辑苗

Fig.4 PCR amplification results of gene-edited seedlings Note: M: DNA marker of D2000; 1-9 are gene edited seedlings obtained from SG10592; 10-21 are gene edited seedlings obtained from SG10610

图5 基因编辑植株获取过程注:A:划线法活化的农杆菌;B:甜瓜种子接种均得到的无菌苗;C:预培养的甜瓜子叶;D:抗潮霉素筛选的芽;E:基因编辑芽在芽伸长培养基上伸长;F:生根培养基中16 d左右的不定芽根;G:移栽土壤的基因编辑苗;H:移栽15 d的基因编辑苗;I:基因编辑植株在人工气候室培养

Fig.5 Diagram of the entire process of obtaining gene edited plants Note: A: Agrobacterium activated by streak method; B: Aseptic seedlings obtained through inoculation of melon seeds; C: Cultivated sweet melon cotyledons; D: Buds screened for resistance to hygromycin; E: Genome editing buds elongate on bud elongation medium; F: Adventitious shoot roots in rooting culture medium for about 16 days; G: Genome editing seedlings of transplanted soil; H: Genome editing seedlings transplanted for 15 days; I: Genome editing plants were cultured in an artificial climate chamber

参考文献 23

[1]	Garcia-Mas J, Benjak A, Sanseverino W, et al. The genome of melon (Cucumis melo L.)[J]. Proceedings of the National Academy of Sciences of the United States of America, 2012, 109(29): 11872-11877. DOI PMID
[2]	池慧芳, 翟丽丽, 刘丽娜, 等. 甜瓜组织培养研究进展及应用概况[J]. 内蒙古农业科技, 2014, 42(5): 108-109.
	CHI Huifang, Zhai Lili, Liu Lina, et al. Research progress and application prospect of melon tissue culture[J]. Inner Mongolia Agricultural Science and Technology, 2014, 42(5): 108-109.
[3]	Nunez-Palenius H G, Gomez-Lim M, Ochoa-Alejo N, et al. Melon fruits: genetic diversity, physiology, and biotechnology features[J]. Critical Reviews in Biotechnology, 2008, 28(1): 13-55.
[4]	张铁刚, 宁雪飞, 王贤磊, 等. 新疆甜瓜“皇后” 再生体系的建立[J]. 北方园艺, 2012, (15): 122-125.
	ZHANG Tiegang, NING Xuefei, WANG Xianlei, et al. Establishment of regeneration system for Cucumis melo L.cv.'Queen' of Xinjiang melon[J]. Northern Horticulture, 2012, (15): 122-125
[5]	M. Galperin, L. Patlis, A. Ovdia, 等. 自交系BU-21/3——甜瓜(Cucumis meloL.)转基因育种的有效工具[J]. 中国西瓜甜瓜, 2003, (4): 26-27.
	M. Galperin, L. Patlis, A. Ovdia, et al. Effective tools for genetically modified breeding[J]. Foreign Watermelon and Melon Technology, 2003, (4):26-27.
[6]	Puonti-Kaerlas J. Cassava biotechnology[J]. Biotechnology and Genetic Engineering Reviews, 1998, 15(1): 329-364.
[7]	王平勇, 徐永阳, 赵光伟, 等. 发根农杆菌介导甜瓜转基因过表达体系的建立[J]. 中国瓜菜, 2019, 32(12): 15-18, 30.
	WANG Pingyong, XU Yongyang, ZHAO Guangwei, et al. Establishment of Agrobacterium rhizogenes-mediated gene overexpression system in melon[J]. China Cucurbits and Vegetables, 2019, 32(12): 15-18, 30.
[8]	Potrykus I. Gene transfer methods for plants and cell cultures[C]//Ciba Foundation Symposium 154‐Bioactive Compounds from Plants: Bioactive Compounds from Plants: Ciba Foundation Symposium 154. Chichester, UK: John Wiley & Sons, Ltd., 2007: 198-212.
[9]	Fang G, Grumet R. Agrobacterium tumefaciens mediated transformation and regeneration of muskmelon plants[J]. Plant Cell Reports, 1990, 9(3): 160-164. DOI PMID
[10]	田红梅, 孙丽萍, 孔庆国. 厚皮甜瓜高效再生体系及遗传转化体系的研究[C]//.中国园艺学会第七届青年学术讨论会论文集, 2006:528-531.
	TIAN Hongmei, SUN Liping, KONG Qingguo, et al. Study on Efficient Regeneration System and Genetic Transformation System of Thick Skin Melon[C]. Proceedings of the 7th Youth Academic Symposium of the Chinese Horticultural Society, 2006:528-531.
[11]	王雪. CRISPR-Cas系统对老汉瓜ACC合成酶基因定点敲除及功能验证[D]. 乌鲁木齐: 新疆大学, 2017.
	WANG Xue. Knocking Out ACC Synthase Gene by CRISPR-Cas System and Verifying the Function in “Laohan” Melon[D]. Urumqi: Xinjiang University, 2017.
[12]	魏爱民, 杜胜利, 韩毅科, 等. 黄瓜转基因技术体系及相关基因转化研究进展[J]. 长江蔬菜, 2010, (20): 4-8.
	WEI Aimin, DU Shengli, HAN Yike, et al. Progress in cucumber transformation techniques and the related genes[J]. Journal of Changjiang Vegetables, 2010, (20): 4-8.
[13]	Nu?ez-Palenius H G, Cantliffe D J, Huber D J, et al. Transformation of a muskmelon ‘Galia’ hybrid parental line (Cucumis melo L. var. reticulatus Ser.) with an antisense ACC oxidase gene[J]. Plant Cell Reports, 2006, 25(3): 198-205.
[14]	何丹. RNAi介导抗WMV、ZYMV、CMV转基因甜瓜材料的研究[D]. 乌鲁木齐: 新疆农业大学, 2016.
	HE Dan. Studying on RNA mediated to resistgance WMV, ZYMV in transgenic melon materials[D]. Urumqi: Xinjiang Agricultural University, 2016.
[15]	李建勇, 丁淑丽, 孙利祥, 等. 影响西瓜农杆菌介导的高效遗传转化效率的主要因子[J]. 浙江农业学报, 2007, 19(3): 197-201.
	LI Jianyong, DING Shuli, SUN Lixiang, et al. Establishment of efficient transgenic system mediated by Agrobacterium tumefaciens in watermelon[J]. Acta Agriculturae Zhejiangensis, 2007, 19(3): 197-201.
[16]	Bezirganoglu I, Hwang S Y, Shaw J F, et al. Efficient production of transgenic melon via Agrobacterium-mediated transformation[J]. Genetics and Molecular Research: GMR, 2014, 13(2): 3218-3227. DOI PMID
[17]	栾非时, 白晶, 朱子成, 等. 农杆菌介导薄皮甜瓜遗传转化体系建立[J]. 东北农业大学学报, 2019, 50(1): 11-18.
	LUAN Feishi, BAI Jing, ZHU Zicheng, et al. Establishment of Agrobacterium mediated genetic transformation system of muskmelon[J]. Journal of Northeast Agricultural University, 2019, 50(1): 11-18.
[18]	方丽, 任海英, 茹水江, 等. 根癌农杆菌介导的甜瓜遗传转化[J]. 浙江农业学报, 2009, 21(3): 211-214.
	FANG Li, REN Haiying, RU Shuijiang, et al. Agrobacterum tumefaciens-mediated genetic transformation in melon[J]. Acta Agriculturae Zhejiangensis, 2009, 21(3): 211-214.
[19]	许娜, 储俊, 夏海武, 等. 黄瓜‘新津春四号’遗传转化体系的建立[J]. 热带亚热带植物学报, 2010, 18(6): 679-684.
	XU Na, CHU Jun, XIA Haiwu, et al. Establishment of genetic transformation system of cucumber ‘new Jinchun 4’[J]. Journal of Tropical and Subtropical Botany, 2010, 18(6): 679-684.
[20]	颜雪, 赵惠新, 王贤磊, 等. 甜瓜抗枯萎病基因表达载体的构建及其转化[J]. 生物技术, 2008, 18(3): 9-11.
	YAN Xue, ZHAO Huixin, WANG Xianlei, et al. Construction of expression vector with R-fom-2 gene and its genetic transformation in melon[J]. Biotechnology, 2008, 18(3): 9-11.
[21]	夏雪琴. 甜瓜CmHSP83基因克隆与遗传转化[D]. 武汉: 华中农业大学, 2015.
	XIA Xueqin. Cloningofthemelon’ CmHSP83 Gene and Genetic Transformation[D]. Wuhan: Huazhong Agricultural University, 2015.
[22]	常尚连. 西瓜糖代谢及甜瓜酸性转化酶反义基因的西瓜遗传转化[D]. 泰安: 山东农业大学, 2006.
	CHANG Shanglian. Sugar Metabolism and Watermelon Genetic Transformation of Acid Invertase Antisense Gene of Melon[D]. Taian: Shandong Agricultural University, 2006.
[23]	王爽. ‘羊角蜜’甜瓜再生与遗传转化体系的建立[D]. 哈尔滨: 东北农业大学, 2021.
	WANG Shuang. Establishment of Plant Regenerationand Genetic Transformation System for Melon ‘16SB29’[D]. Harbin: Northeast Agricultural University, 2019.

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不同甜瓜品种遗传转化再生体系的建立与基因编辑植株的快速获取

Establishment of genetic transformation and regeneration systems for different melon varieties and rapid acquisition of gene edited plants

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