转1174AALdico-2+CTP耐草甘膦优异棉花种质系的创制及其特性

doi:10.3864/j.issn.0578-1752.2023.17.003

Abstract

Abstract:

【Objective】 Weeds are one of the factors limiting cotton growth in cotton production, which not only competes with cotton for nutrition, water, and light, affecting the growth and development of cotton but also the yield and quality of cotton. The aim of this study is to develop excellent cotton germplasms with high glyphosate tolerance by genetic engineering and provide technical support for the commercialization of glyphosate-tolerant cotton to realize chemical weed control and enhance the economic benefits of cotton production. 【Method】 To construct the cotton transformation vector, the EPSPS gene 1174AALdico-2 from Deinococcus radiodurans was connected to the chloroplast transit peptide (CTP), 35S was used as the promoter, and the two target genes were linked in series. The target gene was transformed into the CCRI 49 by Agrobacterium tumefaciens in vivo transformation, and the positive transformants were obtained. The positive transgenic plants obtained were evaluated for glyphosate tolerance, molecular characterization, agronomic and economic traits, in order to obtain outstanding transgenic cotton germplasms with excellent glyphosate tolerance, using the recipient cultivar, CCRI 49, and the non-transgenic NON isolated from the transformants during selfing as the controls. 【Result】 138 positive transformants were obtained by Agrobacterium tumefaciens in vivo transformation. All the transformants were tested for target gene PCR, Southern blot, Western blot, and ELISA detection etc., and 17 positive transformants with clear molecular characteristics and high expression of foreign genes were identified. Southern blot and nucleotide sequencing results revealed that there was great variation in insertion site and copy numbers among the 17 transformants. Among them, ZD131, ZD185, and ZD207 had single-copy insertion sites, and the foreign genes were located on D7, D13, and A12, respectively. The results of glyphosate tolerance identification revealed that the glyphosate tolerance of the three transformants, ZD131, ZD185, and ZD207, was stably inherited across three generations, and the glyphosate tolerance of the transformants was strong, with the ability to tolerate four times the recommended dose of glyphosate in the field. The agronomic and economic features assessment findings revealed that the ZD131, ZD185, and ZD207 grew properly and had excellent agronomic traits such as large boll, high lint percentage, and high boll setting, as well as their lint yield was higher than that of the receptor cultivar control and their fiber quality reached to the high-quality cotton level. 【Conclusion】 The EPSPS gene 1174AALdico-2 from Deinococcus radiodurans connected with the chloroplast transit peptide, and the two gene tandem together was transferred into CCRI 49 by Agrobacterium tumefaciens in vivo transformation technology. After a series of screens, three outstanding transgenic glyphosate-tolerant germplasms, ZD131, ZD185, and ZD207, were obtained. This method not only improves cotton's glyphosate tolerance but also improves the agronomic and economic aspects of transgenic materials.

Key words: cotton, EPSPS, chloroplast transit peptide, glyphosate, lint yield, fiber quality

WANG WanRu, CAO YueFen, SHENG Kuang, CHEN JinHong, ZHAO TianLun, ZHU ShuiJin. The Creation and Characteristics of Cotton Germplasm Lines Transgenic 1174AALdico-2+CTP Gene with Excellent Glyphosate Tolerance[J].Scientia Agricultura Sinica, 2023, 56(17): 3261-3276.

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引物 Primers	序列 Sequence (5′-3′)	退火温度 Annealing temperature (℃)	用途 Purpose
LB-SP1	TTTCTCCATAATAATGTGTGAGTAGTTCCC	68	特异性引物 Special primer
LB-SP2	CTCATGTGTTGAGCATATAAGAAACCCTTAG	68
LB-SP3	CTAAAACCAAAATCCAGTACTAAAATCC	68
Rb-0b	CGTGACTGGGAAAACCCTGGCGTT	68
Rb-1b	CCCAACTTAATCGCCTTGCAGCACATC	68
Rb-2b	GAAGAGGCCCGCACCGATCGCCCTT	68
FP1	GTAATACGACTCACTATAGGGCACGCGTGGTNTCGASTWTSGWGTT	62	融合兼并引物 Fusion arbitrary degenerates primer
FP2	GTAATACGACTCACTATAGGGCACGCGTGGTNGTCGASWGANAWGAA	62
FP3	GTAATACGACTCACTATAGGGCACGCGTGGTWGTGNAGWANCANAGA	62
FP4	GTAATACGACTCACTATAGGGCACGCGTGGTAGWGNAGWANCAWAGG	62
FP5	GTAATACGACTCACTATAGGGCACGCGTGGTNGTAWAASGTNTSCAA	62
FP6	GTAATACGACTCACTATAGGGCACGCGTGGTNGACGASWGANAWGAC	62
FP7	GTAATACGACTCACTATAGGGCACGCGTGGTNGACGASWGANAWGAA	62
FP8	GTAATACGACTCACTATAGGGCACGCGTGGTGTNCGASWCANAWGTT	62
FP9	GTAATACGACTCACTATAGGGCACGCGTGGTNCAGCTWSCTNTSCTT	62
FSP1	GTAATACGACTCACTATAGGGC	62
FSP2	ACTATAGGGCACGCGTGGT	62

供试材料 Plant material	1174AALdico-2±SD (µg·g^-1)
供试材料 Plant material	根Root	茎Stem	叶Leaf	种子Seed
ZD131	14.07±1.22	14.20±1.20	14.22±1.20	12.16±1.11
ZD185	13.87±1.32	13.88±1.40	14.12±1.21	11.26±1.21
ZD207	13.57±1.24	14.22±1.50	13.18±1.30	11.56±1.21
NON (CK1)	0	0	0	0
中棉所49 CCRI 49 (CK2)	0	0	0	0

供试材料 Plant material	IC₅₀草甘膦浓度 IC₅₀ glyphosate concentration (mmol·L^-1)	标准误 Standard error
ZD131	57.60	±2.03a
ZD135	51.40	±2.33ab
ZD157	45.50	±2.34ab
ZD159	44.30	±2.12b
ZD163	47.40	±2.29ab
ZD169	46.60	±2.29ab
ZD171	41.10	±2.33b
ZD185	58.50	±1.78a
ZD187	46.60	±1.75ab
ZD197	39.30	±1.67b
ZD199	38.30	±1.77b
ZD207	58.80	±1.98a
ZD237	45.50	±2.28ab
ZD241	46.30	±1.65ab
ZD243	45.50	±2.56ab
ZD251	50.10	±2.67ab
ZD255	55.30	±2.67a
NON (CK1)	0.33	±0.03c
中棉所49 CCRI 49 (CK2)	0.41	±0.03c

材料 Plant material	推荐剂量（1﹕200稀释） Recommended dose (1﹕200 dilution)					2倍推荐剂量（1﹕100稀释） 2 times recommended dose (1﹕100 dilution)					4倍推荐剂量（1﹕50稀释） 4 times recommended dose (1﹕50 dilution)
材料 Plant material	Ⅰ	Ⅱ	Ⅲ	Ⅳ	平均Average	Ⅰ	Ⅱ	Ⅲ	Ⅳ	平均 Average	Ⅰ	Ⅱ	Ⅲ	Ⅳ	平均 Average
ZD131	1.20	0.20	1.00	0.20	0.65	0.10	1.20	1.40	1.20	0.98	4.80	4.80	8.40	4.80	5.70
ZD185	0.30	1.20	0.50	0.03	0.51	1.40	1.30	0.20	2.10	1.25	9.70	8.60	8.70	6.70	8.43
ZD207	0.00	0.40	0.00	0.20	0.15	1.00	0.60	1.20	0.93	0.93	5.70	3.70	8.30	7.80	6.38
17个转化体平均Average (17)	2.93	3.20	3.24	3.06	3.11	5.11	6.00	6.33	5.90	5.84	17.41	18.68	20.22	19.10	18.85
NON (CK1)	/	/	/	/	/	/	/	/	/	/	/	/	/	/	/
中棉所49 CCRI 49 (CK2)	/	/	/	/	/	/	/	/	/	/	/	/	/	/	/

供试材料 Plant material	平均值Average			标准差SD			变异系数CV (%)
供试材料 Plant material	T₂	T₃	T₄	T₂	T₃	T₄	T₂	T₃	T₄
ZD131	1.01	0.99	0.98	0.02	0.03	0.02	1.98	3.03	2.04
ZD185	1.28	1.26	1.25	0.10	0.10	0.10	7.81	7.94	8.00
ZD207	1.02	1.12	0.93	0.12	0.11	0.09	11.76	9.82	9.68
17个转化体平均Average (17)	5.88	5.82	5.84	0.26	0.31	0.32	4.42	5.33	5.48
NON (CK1)	/	/	/	/	/	/	/	/	/
中棉所49 CCRI 49 (CK2)	/	/	/	/	/	/	/	/	/

The Creation and Characteristics of Cotton Germplasm Lines Transgenic 1174AALdico-2+CTP Gene with Excellent Glyphosate Tolerance

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材料 Plant meterial	T₂					T₃					T₄
材料 Plant meterial	Ⅰ	Ⅱ	Ⅲ	Ⅳ	平均 Average	Ⅰ	Ⅱ	Ⅲ	Ⅳ	平均 Average	Ⅰ	Ⅱ	Ⅲ	Ⅳ	平均 Average
ZD131	100	100	100	100	100	100	100	100	100	100	100	100	100	100	100
ZD185	100	100	100	100	100	100	100	100	100	100	100	100	100	100	100
ZD207	100	100	100	100	100	100	100	100	100	100	100	100	100	100	100
17个转化体平均Average (17)	100	100	100	100	100	100	100	100	100	100	100	100	100	100	100
NON (CK1)	/	/	/	/	/	/	/	/	/	/	/	/	/	/	/
中棉所49 CCRI 49 (CK2)	/	/	/	/	/	/	/	/	/	/	/	/	/	/	/

供试材料 Plant material	生育期 Growth period (d)	株高 Plant height (cm)	果枝数 Fruit branches	单株铃数 Bolls per plant	铃重 Boll weight (g)	衣分 Lint percentage (%)	枯萎病指 Fusarium wilt index
供试材料 Plant material	生育期 Growth period (d)	株高 Plant height (cm)	果枝数 Fruit branches	单株铃数 Bolls per plant	铃重 Boll weight (g)	衣分 Lint percentage (%)	发病率 Disease rate (%)	病指 Disease index
ZD131	120	119.7	16.2	26.4	5.8	42.5	24.3	7.8
ZD185	125	115.0	12.3	22.4	5.4	40.1	29.0	11.8
ZD207	129	98.6	12.7	25.6	5.2	40.5	29.3	11.2
17个转化体平均Average (17)	129	98.6	12.7	21.1	5.2	40.5	29.3	11.2
NON (CK1)	136	124.4	16.8	22.1	5.6	38.4	27.8	15.9
中棉所49 CCRI 49 (CK2)	126	126.1	17.7	21.7	5.5	39.0	34.8	20.5

供试材料 Plant material	籽棉Seed cotton			皮棉Lint
供试材料 Plant material	产量 Yield (kg·hm^-2)	为CK1 as CK1 (%)	为CK2 as CK2 (%)	产量 Yield (kg·hm^-2)	为CK1 as CK1 (%)	为CK2 as CK2 (%)
ZD131	6092.54	107.7	106.9	2589.55	119.2**	116.5**
ZD185	5865.67	103.7	102.9	2352.24	108.3*	105.8
ZD207	5617.91	99.3	98.6	2274.63	104.8	102.4
17个转化体平均Average (17)	5105.97	90.3	89.6	2068.66	95.2	93.0
NON (CK1)	5656.72	100.0	99.3	2171.64	100.0	97.7
中棉所49 CCRI 49 (CK2)	5698.51	100.7	100.0	2222.39	102.3	100.0

供试材料 Plant material	上半部长度 Fiber length (mm)	整齐度指数 Uniformity (%)	断裂比强度 Strength (cN·tex^-1)	马克隆值 Micronaire	伸长率 Elongation (%)
ZD131	32.7	85.7	32.1	4.9	6.8
ZD185	30.7	85.3	30.3	5.0	6.8
ZD207	31.3	85.4	34.6	4.5	6.7
17个转化体平均Average (17)	28.0	82.9	28.9	5.3	6.8
NON (CK1)	32.3	85.5	30.4	5.0	6.9
中棉所49 CCRI 49 (CK2)	33.1	82.3	31.2	4.2	6.6

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