耐除草剂棉花GGK2的遗传稳定性分析及性状鉴定

doi:10.3864/j.issn.0578-1752.2023.17.002

摘要/Abstract

摘要： 【目的】 明确转基因耐除草剂棉花GGK2的遗传稳定性和营养品质等性状，为其产业化应用提供数据基础和技术支撑。 【方法】 针对实验室培育的新型耐除草剂棉花GGK2，选取其T₃、T₄、T₅代开展试验研究。利用特异性PCR和Southern blot开展基因组整合稳定性分析；利用实时荧光定量PCR、ELISA和胶体金试纸条开展表达稳定性分析；通过田间喷施草甘膦测试其除草剂耐受稳定性，以及依据相关国际或国内行业标准进行棉籽营养成分分析等。 【结果】 抗除草剂基因GR79 EPSPS和GAT，以及筛选标记基因NPTⅡ以单拷贝形式整合到棉花GGK2基因组D10染色体，且能够在T₃、T₄、T₅代转基因棉花中稳定遗传，同时发现基因组中不含有遗传转化载体的骨架片段。表达分析发现GR79 EPSPS、GAT和NPTⅡ 3个基因在棉花GGK2的不同时期、不同组织部位均有表达，其中，叶片中的表达量相对较高。四叶期、盛花期、吐絮期3个蛋白在叶片中表达量分别介于128.7—192.4 µg·g^-1、24.4—35.0 µg·g^-1和17.0—23.9 µg·g^-1鲜重，利用GR79 EPSPS单克隆抗体制备的胶体金试纸条可以对田间棉花GGK2叶片实现快速鉴定。除草剂耐受性分析结果显示，转基因棉花GGK2可耐受4倍生产使用剂量的草甘膦除草剂。T₃—T₅不同世代棉花喷施除草剂后均未发现受害症状，而且整个生育期的生长发育未受影响。棉籽营养成分分析表明，来源于新疆、河北和河南种植的GGK2棉籽中，水分、灰分、蛋白质、脂肪、粗纤维、维生素及脂肪酸含量与非转基因对照之间无显著差异，各指标的含量水平均位于ILSI数据库报道的范围内。 【结论】 转基因棉花新种质GGK2的耐除草剂性状遗传稳定性好、除草剂耐受能力强、籽粒营养成分与非转基因对照之间无显著差异，在耐除草剂棉花生物育种具有重要的应用价值且产业化前景好。

关键词: 棉花, GGK2, 耐除草剂, 草甘膦, 遗传稳定性

Abstract:

【Objective】The objective of this study is to confirm the target traits and genetic stability of transgenic glyphosate- resistant cotton GGK2 and provide technical support for its commercialization. 【Method】T₃, T₄, and T₅ transgenic cotton plants GGK2 were subjected to insertion site-specific PCR, Southern blot, ELISA, bioassays in the laboratory and field, analysis of target herbicide tolerance, and investigation of nutritional constituents. 【Result】The results indicated that the target genes, GR79 EPSPS and GAT, were integrated into the cotton genome as single copies and stably inherited in GGK2 plants. In GGK2 cotton, GR79 EPSPS, GAT, and NPTⅡ proteins were expressed at different stages and in different tissues, with relatively high expression levels in the leaves. At the four-leaf stage, bud stage and boll opening stag, the expression levels in leaves were 128.7-192.4 µg·g^-1, 24.4-35.0 µg·g^-1, and 17.0-23.9 µg·g^-1 fresh weight for GR79 EPSPS, GAT, and NPTⅡ, respectively. In the field, transgenic cotton GGK2 tolerated up to four times the recommended medium dose of glyphosate application. No significant differences were observed in agronomic traits and nutritional constituents compared to the control, Coker312. 【Conclusion】These data demonstrate that transgenic cotton GGK2 is genetically stable and highly resistant to herbicides. Therefore, it can be utilized for breeding high-glyphosate- resistant commercial cotton varieties.

Key words: cotton, GGK2, herbicide tolerance, glyphosate, genetic stability

梁成真, 臧有义, 孟志刚, 王远, MUBASHIR Abbas, 何海焱, 周琪, 魏云晓, 张锐, 郭三堆. 耐除草剂棉花GGK2的遗传稳定性分析及性状鉴定[J]. 中国农业科学, 2023, 56(17): 3251-3260.

LIANG ChengZhen, ZANG YouYi, MENG ZhiGang, WANG Yuan, MUBASHIR Abbas, HE HaiYan, ZHOU Qi, WEI YunXiao, ZHANG Rui, GUO SanDui. Identification of Target Traits and Genetic Stability of Transgenic Cotton GGK2[J]. Scientia Agricultura Sinica, 2023, 56(17): 3251-3260.

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处理时间 Time	性状 Phenotype	喷施物 Spray	柯字棉312 Coker312	GGK2
处理时间 Time	性状 Phenotype	喷施物 Spray	柯字棉312 Coker312	T₃	T₄	T₅
喷施后2周 Two weeks after spraying	株高 Plant height (cm)	草甘膦Glyphosate	/	26.74±1.40	27.44±1.74	28.12±1.41
	株高 Plant height (cm)	清水Water	27.34±2.29	27.11±1.08	26.42±1.84	26.72±1.22
	叶片受害率 Leaf damage rate (%)	草甘膦Glyphosate	100.00±0.00	0.96±0.10	0.71±0.23	0.82±0.07
	叶片受害率 Leaf damage rate (%)	清水Water	0.00±0.00	0.00±0.00	0.00±0.00	0.00±0.00
喷施后4周 Four weeks after spraying	株高 Plant height (cm)	草甘膦Glyphosate	/	46.88±2.54	47.03±1.22	47.23±3.18
	株高 Plant height (cm)	清水Water	47.60±1.92	47.27±0.53	46.93±1.52	48.03±1.39
	叶片受害率 Leaf damage rate (%)	草甘膦Glyphosate	100.00±0.00	1.21±0.26	0.83±0.03	1.13±0.11
	叶片受害率 Leaf damage rate (%)	清水Water	0.00±0.00	0.00±0.00	0.00±0.00	0.00±0.00
/：全部死亡 /: All dead

名称Name	水分 Water content (g/100 g)			灰分 Ash content (g/100 g)			蛋白质 Protein content (g/100 g)			脂肪 Fat content (g/100 g)
名称Name	T₃	T₄	T₅	T₃	T₄	T₅	T₃	T₄	T₅	T₃	T₄	T₅
GGK2	9.10	8.57	8.84	4.10	4.20	4.10	20.80	20.80	23.90	17.30	16.60	18.20
柯字棉312 Coker312	8.15	8.77	8.37	4.00	4.10	4.20	22.10	21.10	23.60	17.20	17.10	18.10
	粗纤维 Coarse fiber content (g/100 g)			维生素E <BOLD>V</BOLD>itamin E content (mg/100 g)			维生素B2 <BOLD>V</BOLD>itamin B2 content (mg/100 g)			肉豆蔻酸 Myristic chemicing (%)
	T₃	T₄	T₅	T₃	T₄	T₅	T₃	T₄	T₅	T₃	T₄	T₅
GGK2	14.40	14.60	17.80	8.48	7.84	7.09	5.96×10^-2	5.85×10^-2	5.68×10^-2	0.59	0.60	0.82
柯字棉312 Coker312	15.50	17.00	15.70	9.71	10.30	13.20	5.75×10^-2	5.57×10^-2	5.65×10^-2	0.69	0.69	0.70
	棕榈酸 Palmitic acid (%)			棕榈油酸 Palmitoleic acid (%)			十七烷酸 Heptacosanoic acid (%)			硬脂酸 Stearine acid (%)
	T₃	T₄	T₅	T₃	T₄	T₅	T₃	T₄	T₅	T₃	T₄	T₅
GGK2	21.30	21.40	25.40	0.64	0.64	0.75	7.39×10^-2	7.56×10^-2	8.17×10^-2	2.20	2.22	2.56
柯字棉312 Coker312	22.10	22.00	22.10	0.61	0.61	0.61	7.91×10^-2	7.80×10^-2	7.85×10^-2	2.35	2.41	2.37
	油酸 Oleic acid (%)			亚油酸 Linoleic acid (%)			十九烷酸 Nonadecanoic acid (%)			亚麻酸 Linolenic acid (%)
	T₃	T₄	T₅	T₃	T₄	T₅	T₃	T₄	T₅	T₃	T₄	T₅
GGK2	15.70	15.30	14.60	57.50	57.80	52.60	0.23	0.24	0.18	0.23	0.22	0.16
柯字棉312 Coker312	14.70	14.70	14.60	57.50	57.50	57.60	0.25	0.28	0.26	0.18	0.19	0.18
	花生酸 Arachic acid (%)			山嵛酸 Behenic acid (%)			棉酚 Gossypol (mg·kg^-1)			植酸酶 Phytase (g·kg^-1)
	T₃	T₄	T₅	T₃	T₄	T₅	T₃	T₄	T₅	T₃	T₄	T₅
GGK2	0.20	0.19	0.26	0.13	0.13	0.13	5.42×10³	5.16×10³	5.45×10³	28.50	29.10	29.10
柯字棉312 Coker312	0.23	0.22	0.22	0.14	0.14	0.14	5.41×10³	5.25×10³	5.39×10³	30.00	26.40	27.70