昼夜变温下高温与干旱胁迫对Bt棉毒蛋白含量的影响及其生理机制

doi:10.3864/j.issn.0578-1752.2022.23.004

摘要/Abstract

摘要： 目的明确Bt棉叶片Cry1Ac毒蛋白含量对昼夜变温下高温干旱胁迫响应及其生理机制，为生产中Bt棉抗虫性的安全稳定利用提供参考。方法 2019—2020年在扬州大学农学院，以常规种泗抗1号（SK-1）和杂交种泗抗3号（SK-3）为材料，以温度和土壤水分含量为因子，温度分别设为34℃（白天，7：00—19：00）/28℃（夜间，19：00—7：00）（A1）、38℃/28℃（A2）；土壤水分含量分别为田间土壤最大持水量的50%（B1）和60%（B2），并以32℃/28℃、田间土壤最大持水量的75%为对照（CK）。各处理分别持续4、7、10 d（DAS）。结果不同处理导致叶片中Cry1Ac毒蛋白含量降低，且随着胁迫时间的延长，下降幅度增加。处理间相比，A1B2处理下降幅度最少，7 DAS后开始显著低于CK；A1B1处理下降幅度其次，4 DAS后显著低于CK；A2B1、A2B2处理在4 DAS显著下降。可溶性蛋白（SP）含量、硝酸还原酶（NR）、谷氨酸丙酮酸转氨酶（GPT）、谷氨酸草酰乙酸转氨酶（GOT）、谷氨酰胺合成酶（GS）、游离氨基酸（aa）和谷氨酸合成酶（GOGAT）等活性变化趋势与毒蛋白一致，且呈极显著正相关，而Bt基因表达量、单宁含量、蛋白酶、肽酶活性则呈上升趋势。逐步回归和通径分析筛选出NR、GPT、GS等3个关键指标可反映Bt棉Cry1Ac毒蛋白含量高低，且三者对Cry1Ac毒蛋白含量有较大的正效应。结论昼夜变温下高温和干旱互作导致Bt棉Cry1Ac毒蛋白含量降低，且随持续期延长下降幅度逐渐增大。其中34℃/28℃和田间土壤最大持水量60%胁迫7—10 d内与对照无显著差异。但与昼夜持续高温相比，昼夜变温的高温胁迫下Cry1Ac毒蛋白含量下降幅度减小和时期明显推迟。NR、GPT、GS是决定Cry1Ac毒蛋白含量高低的关键指标。

关键词: Bt棉, Cry1Ac毒蛋白, 高温, 干旱, 生理机制

Abstract:

【Objective】 This study was conducted to investigate the effects of alternating high temperature and drought on the content of Cry1Ac protein in Bt cotton and the underlying physiological mechanism, so as to provide a reference for the safe and stable utilization of insect resistance of Bt cotton in production.【Method】 The conventional cultivar Sikang 1 (SK-1) and hybrid cultivar Sikang 3 (SK-3) were used in 2019 and 2020 in experimentally controlled greenhouse, Yangzhou University. From 7:00 am to 7:00 pm, two high-temperature treatments [34℃ (A1) and 38℃ (A2)] were imposed on cotton plants, followed by an optimum temperature 28℃ during the remaining night hours. There were two treatments for soil moisture content, which were 50% (B1) and 60% (B2) field capacity. The treatment with 32℃/28℃ and 75% field capacity was set as the control (CK). The leaf Cry1Ac protein content and its physiological mechanism were detected on 4, 7, and 10 days after stress (DAS), respectively.【Result】 Compared with CK, the Cry1Ac protein content all decreased under the stresses of high temperature and drought, and with the extension of the stress time, greater decrease was observed. The extent of decline for A1B2 was the smallest, followed by A1B1, while A2B1 and A2B1 were the largest. The Cry1Ac protein content under A1B2 was significantly lower than CK after 7 DAS, while the significant differences between A1B1, A2B2, A2B2 and CK were detected after 4 DAS. The Bt gene expression level, soluble protein (SP), free amino acid (aa) contents, nitrate reductase (NR), the glutamic pyruvic transaminase (GPT), glutamic oxaloacetate transaminase (GOT), glutamine synthetase (GS) and glutamate synthase (GOGAT) activities showed a downward trend. While the tannin content, the activities of protease and peptidase showed an upward trend. The correlation analysis and path analysis showed SP, aa, NR, GPT, GOT, GS, and GOGAT were positively correlated with Cry1Ac protein content. The tannin content, activities of protease, and peptidase were negatively correlated with Cry1Ac protein content. NR, GPT and GS could be key indices for the Cry1Ac protein content.【Conclusion】 The interaction of high temperature and drought resulted in the decrease of Cry1Ac protein content in Bt cotton, with greater decrease observed as the stress extended. There was no significant difference between the treatment with 34℃/28℃ and 60% field capacity and CK in 7-10 DAS. The reduction extent of Cry1Ac for alternating high temperature regime and drought decreased and the period was delayed. NR, GPT and GS could be key indices for the Cry1Ac protein content.

Key words: Bt cotton, Cry1Ac protein, high temperature, drought, physiological mechanism

尹彦雨, 邢雨桐, 吴天凡, 王李妍, 赵子胥, 胡天然, 陈源, 陈媛, 陈德华, 张祥. 昼夜变温下高温与干旱胁迫对Bt棉毒蛋白含量的影响及其生理机制[J]. 中国农业科学, 2022, 55(23): 4614-4625.

YIN YanYu, XING YuTong, WU TianFan, WANG LiYan, ZHAO ZiXu, HU TianRan, CHEN Yuan, CHEN Yuan, CHEN DeHua, ZHANG Xiang. Cry1Ac Protein Content Responses to Alternating High Temperature Regime and Drought and Its Physiological Mechanism in Bt Cotton[J]. Scientia Agricultura Sinica, 2022, 55(23): 4614-4625.

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年份 Year	品种 Cultivar	处理 Treatment	胁迫后天数Days after stress (DAS)
年份 Year	品种 Cultivar	处理 Treatment	4 d	7 d	10 d
2019	SK-1	CK	894.29±5.56a	670.72±2.62a	558.93±16.4a
		A1B1	422.30±1.60abc	345.67±82.45bc	227.52±42.32b
		A1B2	598.00±4.66ab	438.14±3.77b	434.73±34.66a
		A2B1	111.75±1.45c	83.81±1.45d	60.16±9.53c
		A2B2	274.57±2.66bc	219.62±9.61cd	181.69±2.82bc
2020	SK-1	CK	835.63±14.62ab	826.02±5.32a	810.17±0.34b
		A1B1	424.98±40.69def	343.43±52.67de	294.50±1.93f
		A1B2	654.28±26.05abcd	387.93±4.16cde	366.11±2.79e
		A2B1	104.20±2.40g	93.39±2.04g	33.62±2.06i
		A2B2	255.69±2.02fg	214.81±1.70f	114.88±0.66h
	SK-3	CK	873.93±16.69a	859.89±17.97a	892.94±3.23a
		A1B1	606.27±60.58bcde	502.56±58.28bc	464.49±1.64d
		A1B2	705.66±25.49abc	605.46±58.32b	523.92±4.19c
		A2B1	370.71±3.29ef	313.82±2.61ef	282.39±1.88g
		A2B2	481.85±1.99cdef	457.19±1.64cd	368.96±7.99e

品种 Cultivar	处理 Treatment	胁迫后天数Days after stress (DAS)
品种 Cultivar	处理 Treatment	4 d	7 d	10 d
SK-1	CK	3.15±0.20c	3.81±0.25d	4.04±0.01ef
	A1B1	3.84±0.09bc	4.84±0.61c	5.38±0.14c
	A1B2	3.17±0.02c	3.95±0.04d	4.96±0.04cd
	A2B1	5.50±1.03a	6.17±0.19a	8.01±1.18a
	A2B2	4.49±0.50b	5.45±0.24b	6.73±0.61b
SK-3	CK	0.73±0.13g	0.77±0.07g	1.15±0.06h
	A1B1	1.76±0.22de	1.94±0.05f	3.87±0.07f
	A1B2	1.02±0.11fg	1.53±0.01f	1.77±0.04h
	A2B1	2.25±0.31d	2.51±0.36e	4.61±0.30de
	A2B2	1.51±0.47ef	1.82±0.22f	2.80±0.07g

年份 Year	品种 Cultivar	处理 Treatment	SP含量 SP content			aa含量 aa content
年份 Year	品种 Cultivar	处理 Treatment	4 DAS	7 DAS	10 DAS	4 DAS	7 DAS	10 DAS
2019	SK-1	CK	2.60±0.20a	2.02±0.05a	1.14±0.05a	1.50±0.10a	1.05±0.04a	0.91±0.02a
		A1B1	1.42±0.00bc	0.97±0.04c	0.67±0.03c	0.98±0.07b	0.82±0.05bc	0.64±0.04c
		A1B2	2.21±0.11ab	1.68±0.23b	0.97±0.02b	1.15±0.20b	0.90±0.08ab	0.70±0.00b
		A2B1	0.72±0.01c	0.36±0.05e	0.34±0.02e	0.67±0.02c	0.54±0.01d	0.53±0.03d
		A2B2	1.34±0.08bc	0.62±0.01d	0.55±0.06d	0.95±0.01b	0.69±0.16cd	0.54±0.03d
2020	SK-1	CK	5.36±0.19b	4.42±0.10b	2.5±0.19b	1.81±0.47a	1.37±0.06a	1.06±0.04a
		A1B1	4.70±0.13cd	3.35±0.03e	1.17±0.02f	1.22±0.11b	1.03±0.10c	0.81±0.02c
		A1B2	5.03±0.14bc	3.76±0.07d	1.74±0.08d	1.42±0.08ab	1.18±0.08b	0.92±0.02b
		A2B1	3.57±0.22f	2.23±0.08h	0.63±0.02h	0.69±0.04c	0.68±0.01d	0.62±0.04e
		A2B2	4.20±0.17e	2.99±0.30f	0.95±0.04g	1.12±0.04bc	0.92±0.10c	0.72±0.01d
	SK-3	CK	5.81±0.53a	4.73±0.06a	3.08±0.02a	2.19±0.02a	1.85±0.02a	1.50±0.05a
		A1B1	4.83±0.05c	3.36±0.03e	1.34±0.01e	1.88±0.10b	1.34±0.06b	1.01±0.02c
		A1B2	5.27±0.28b	3.98±0.08c	2.11±0.03c	2.06±0.16ab	1.39±0.04b	1.10±0.02b
		A2B1	3.65±0.01f	2.44±0.02g	0.91±0.04g	1.59±0.04c	0.98±0.18c	0.80±0.03e
		A2B2	4.33±0.03de	3.17±0.03ef	1.15±0.03f	1.66±0.12c	1.10±0.07c	0.89±0.04d

年份 Year	品种 Cultivar	处理 Treatment	GPT活性 GPT Activity			GOT活性 GOT Activity
年份 Year	品种 Cultivar	处理 Treatment	4DAS	7DAS	10DAS	4DAS	7DAS	10DAS
2019	SK-1	CK	22.31±3.57a	21.29±0.14a	18.65±0.11a	31.02±5.34a	27.57±2.13a	26.19±0.70a
		A1B1	18.88±0.15bc	16.82±0.80c	10.48±0.25c	25.93±0.21b	23.83±024b	19.95±0.57c
		A1B2	20.57±2.87ab	18.91±0.25b	14.01±0.22b	27.05±2.64ab	26.47±0.63ab	24.57±0.64b
		A2B1	16.56±0.36d	10.24±0.35e	5.42±0.20e	22.33±0.34b	18.45±0.23d	13.03±0.36e
		A2B2	17.41±0.15cd	12.52±0.23d	8.69±0.16d	25.31±0.21b	20.91±0.10c	17.09±0.25d
2020	SK-1	CK	24.05±3.48c	22.81±1.73c	20.64±0.63b	36.55±2.32c	35.14±0.36c	29.84±0.89c
		A1B1	18.65±2.48f	14.81±0.80e	11.10±0.35g	31.71±2.25d	26.20±0.52e	20.19±0.10f
		A1B2	22.70±1.06cd	18.51±0.86d	12.89±0.82f	34.84±1.55c	30.95±1.54d	25.82±0.40e
		A2B1	13.01±0.21h	7.42±0.15g	4.25±0.18j	24.13±0.82f	19.90±0.41f	14.21±0.04g
		A2B2	16.19±0.30g	10.73±0.19f	9.60±0.73i	27.03±0.37e	21.45±0.14f	18.53±0.36f
	SK-3	CK	30.79±0.37a	29.70±0.38a	26.23±0.28a	45.76±1.54a	41.33±1.30a	40.71±0.43a
		A1B1	28.67±0.17b	22.24±1.09c	16.59±0.68d	39.80±2.54b	36.01±1.61c	28.06±1.07d
		A1B2	29.92±1.46ab	24.57±0.84b	19.57±0.63c	43.99±1.38a	38.69±0.93b	35.98±0.67b
		A2B1	20.85±0.08e	15.76±0.54e	10.37±0.36h	31.88±1.38d	26.41±1.25e	19.67±1.67f
		A2B2	22.47±0.29d	18.12±0.58d	15.04±1.18e	34.48±2.14cd	31.81±1.47d	26.28±1.78e

年份 Year	品种 Cultivar	处理 Treatment	GS活性 GS activity (U·g^-1 FW·min^-1)			GOGAT活性 GOGAT activity (μmol·g^-1 FW·min^-1)
年份 Year	品种 Cultivar	处理 Treatment	4DAS	7DAS	10DAS	4DAS	7DAS	10DAS
2019	SK-1	CK	7.35±0.30a	6.61±0.28a	5.95±0.31a	3.36±0.43a	3.19±0.02a	3.03±0.04a
		A1B1	4.96±0.10ab	3.42±0.06bc	3.31±0.45c	2.33±0.00abc	2.01±0.09bc	1.93±0.03b
		A1B2	5.28±0.11ab	4.73±0.09b	3.86±0.04b	2.55±0.15ab	2.27±0.14ab	2.01±0.26b
		A2B1	3.63±0.09b	2.32±0.24c	1.58±0.39e	1.33±0.11c	1.26±0.04c	0.90±0.04c
		A2B2	4.63±0.08b	2.88±0.11c	2.17±0.32d	2.00±0.12bc	1.94±0.04bc	1.74±0.19bc
2020	SK-1	CK	8.68±0.25c	7.17±0.11bc	6.21±0.07b	3.41±0.19d	3.16±0.02c	2.80±0.24d
		A1B1	8.21±0.06d	5.09±0.30de	4.27±0.19e	3.12±0.17e	2.22±0.10f	1.55±0.01f
		A1B2	8.47±0.20cd	6.59±0.40c	5.66±0.32c	3.30±0.10de	2.95±0.24d	2.50±0.15e
		A2B1	5.46±0.32f	3.71±0.17g	1.43±0.29h	1.80±0.15g	1.65±0.07h	0.98±0.02h
		A2B2	6.89±0.10e	4.33±0.29f	2.5±0.35g	2.25±0.05f	1.97±0.08g	1.32±0.03g
	SK-3	CK	10.29±0.21a	9.32±0.44a	7.31±0.25a	4.91±0.09a	4.49±0.17a	4.53±0.11a
		A1B1	9.84±0.09b	7.58±0.68b	5.18±0.07d	4.15±0.08b	3.97±0.17b	3.24±0.02c
		A1B2	9.88±0.09ab	8.74±0.38a	6.49±0.32b	4.76±0.11a	4.34±0.03a	3.55±0.05b
		A2B1	6.87±0.36e	4.87±0.12ef	2.48±0.18g	3.22±0.07de	2.44±0.09e	1.65±0.10f
		A2B2	8.37±0.39cd	5.70±0.10d	3.31±0.15f	3.65±0.02c	3.11±0.03cd	2.36±0.09e