籼稻背景下抑制不同ALK等位基因表达对稻米品质的影响

doi:10.16819/j.1001-7216.2019. 9090

摘要/Abstract

摘要：

目的稻米糊化温度是影响稻米品质的重要指标,该性状受主效基因ALK/SSII-3调控,ALK基因具有多个复等位基因,本研究旨在通过RNAi技术明确籼稻亚种中两个不同ALK等位基因的效应。方法以分别含有ALK^c和ALK^b等位基因的高糊化温度品种珍汕97B和低糊化温度品种龙特甫B为试验材料,使用RNAi技术构建ALK表达下调的转基因株系,通过对其稻米理化品质的测定来明确不同等位基因表达下调对稻米品质的影响。结果对不同转基因水稻目的基因的表达分析显示本研究中转基因株系的ALK基因受到了不同程度的干扰。重点分析了不同RNAi株系稻米的糊化温度,结果表明珍汕97B的RNAi转基因稻米的糊化温度极显著降低,而在低糊化温度品种龙特甫B背景中下调表达ALK基因后对糊化温度的影响较小;转基因株系与未转化亲本相比,米粉的起始糊化温度都显著降低,表现为提前糊化;在珍汕97B背景下干扰系的峰值温度与未转化亲本相比极显著降低,而在龙特甫背景下米粉的峰值温度与未转化对照相比显著降低。对不同转基因系的理化品质分析表明,ALK下调表达植株稻米的表观直链淀粉含量显著增加,下调表达ALK后会引起米粉峰值黏度和崩解值的改变。高糊化温度品种珍汕97B干扰系与未转化对照相比胶稠度呈现极显著性差异,而低糊化温度品种龙特甫干扰系的胶稠度与未转化对照相比没有差异。结论下调表达ALK等位基因对稻米理化品质产生显著影响,并且干扰不同等位基因的效应存在明显差异,即籼稻中的两个ALK等位基因的效应存在显著差异。

关键词: 水稻, ALK等位基因, RNA干扰, 糊化温度, 稻米品质

Abstract:

【Objective】 Gelatinization temperature (GT) is one of the key physicochemical properties in rice quality, which is mainly regulated by ALK (SSII-3) gene. In general, there are two ALK alleles among indica cultivars. To detect their functional differentiation in indica rice,【Method】 Zhenshan 97B (a high GT variety carrying ALK^c allele) and Longtefu B (a low GT variety carrying ALK^b allele), were used as receptors for the generation of transgenic rice with down-regulation of ALK expression by RNA interference (RNAi).【Result】 Down-regulation of ALK gene significantly decreased the GT of the transgenic lines. Due to the difference of original GT between the two receptors, the GT of transgenic rice lines derived from Zhenshan 97B (a high GT variety) decreased significantly, but it is slightly decreased in transgenic plants derived from Longtefu B (a low GT variety). The differential scanning calorimetry (DSC) results showed that the initial temperature of RNAi transgenic rice was significantly lower than the corresponding control and the transgenic lines were gelatinized in advance. The peak value of GT(T_p) in RNAi rice grains was significantly lower than that of the control under Zhenshan 97B background. However, T_p of RNAi rice grains under Longtefu B background was significantly lower than the control to a lesser extent. Also, down-regulation of ALK expression had a significant effect on rice physical-chemical characteristics. An increase of apparent amylose content in RNAi transgenic plants was detected due to the decreased expression of ALK gene. Besides, the pasting properties showed that down-regulation of ALK gene had obvious effects on peak viscosity and breakdown value, improving the taste of the transgenic rice. The gel consistency was significantly different among Zhenshan 97B RNAi lines and their parents, but no difference was found in Longtefu-derived transgenic lines.【Conclusion】 RNA interference to ALK allele expression had a significant effect on rice quality, especially the gelatinization characters. Down-regulated expression level of ALK^c allele would cause larger variation of physical-chemical characteristics between transgenic rice and their parent than that of ALK^b allele.

Key words: rice, ALK allele, RNA interference, gelatinization temperature, rice grain quality

中图分类号:

Q755
S511.01

陈专专, 李先锋, 仲敏, 葛家奇, 范晓磊, 张昌泉, 刘巧泉. 籼稻背景下抑制不同ALK等位基因表达对稻米品质的影响[J]. 中国水稻科学, 2019, 33(6): 513-522.

Zhuanzhuan CHEN, Xianfeng LI, Min ZHONG, Jiaqi GE, Xiaolei FAN, Changquan ZHANG, Qiaoquan LIU. Grain Quality as Affected by Down-regulation of Expression of Different ALK Alleles in indica Rice (Oryza sativa L.)[J]. Chinese Journal OF Rice Science, 2019, 33(6): 513-522.

图/表 7

图1 ALK-RNAi载体结构及转基因水稻的鉴定 A-用引物4342(GC/TT)鉴定亲本对照龙特甫B(LTFB)和珍汕97B(ZS97B)的ALK基因型。B-含ALK-RNAi 结构双元载体的T-DNA区结构。P35S和Tnos, 分别表示CaMV35S基因的启动子和终止子区; NOS-农杆菌胭脂碱合成酶基因终止子; Hyg-潮霉素抗性基因; LB和RB分别表示T-DNA区的左右边界序列; Anti和Sense分别表示目的基因片段的反向和正向结构;图中箭头表示设计引物方向。C-ALK-RNAi转基因水稻植株的PCR鉴定。D-ALK-RNAi转基因水稻植株胚乳中ALK基因表达量分析。L-ALKb-RNAi-1、L-ALKb-RNAi-2和L-ALKb-RNAi-3为龙特甫背景下的ALKb-RNAi转基因纯合系;Z-ALKc-RNAi-2-1、Z-ALKc-RNAi-2-2和Z-ALKc-RNAi-2-3为同时携带有ALKc等位基因和ALKb-RNAi干扰结构的转基因材料纯合系。对不同背景下的转基因系和未转化对照的表达量进行方差分析,并用新复极差法进行多重比较。**表示转基因系与未转化对照之间的差异达0.01显著水平,*表示转基因系与未转化对照之间的差异达0.05显著水平(n=3)。

Fig. 1. ALK-RNAi construction and idendification of transgenic rice. A, ALK genotype in Longtefu B(LTFB) and Zhenshan 97B(ZS97B) on sites 4342 and 4343 based on primer 4342(GC/TT). B, The T-DNA structure of ALK-RNAi construct. P35S and Tnos represent the promoter and terminator region of the CaMV 35S gene respectively; NOS represents agrobacterium nopaline synthetase gene terminator; Hyg represents hygromycin resistance gene; LB and RB represent the left and right boundary sequences of the T-DNA region respectively; the anti-sense and the sense represent the reverse and forward structure of the target gene segment respectively; and the arrow in the figure indicates the direction of the designed primer. C, PCR analysis of ALK-RNAi transgenic rice plants. D, Transcription level expression of ALK Gene in RNAi Line. L-ALKb-RNAi-1, L-ALKb-RNAi-2 and L-ALKb-RNAi-3 are ALKb-RNAi transgenic homozygous lines under the background of Longtefu and Z-ALKc-RNAi-2-1, Z-ALKc-RNAi-2-2 and Z-ALKc-RNAi-2-3 are homozygous transgenic lines carrying both ALKb-RNAi interference structure and ALKc allele. Data were subjected to one-way analysis of variance (ANOVA) depending on the experiment, followed by a comparison of the means according to a Duncan’s multiple range test at P< 0.05 or P < 0.01. Double asterisks denote a highly significant difference between transgenic line and its wild type (P<0.01), single asterisk denotes a significant difference between transgenic line and it’s wild type (0.01≤ P<0.05). (n=3).

表1 本研究所用的PCR引物

Table 1 Primers used in this study.

正向引物 Forward primer	引物序列 Primer sequence(5′-3′)	反向引物 Reverse primer	引物序列 Primer sequence(5′-3′)
ALK-5	GTCCATGGTCGACCTCAAGTAAGAACGGAG	ALK-3	GTACTAGTCGCCTTTGGCTTC
4342(GC/TT)-F1	CAAGGAGAGCTGGAGGGGGC	4342(GC/TT)-R1	CATGCCGCGCACCTGGAAA
4342(GC/TT)-F	TCGGCGGGCTGAGGGACAC	4342(GC/TT)-R	TCGCATCAATGGACATAACAAACAC
INT-F	CCTCGTAATCAATTTTAGAT	NOS-R	GACCGCACAGGATTCAAT
ALK-RT-F	TGATCTGAACGAACCGGACG	ALK-RT-R	TGGGTAAAGCACCTGCAACA

图2 ALK-RNAi转基因稻米的糊化温度 A-ALK-RNAi水稻株系精米在1.7% KOH中的碱消值; B-ALK-RNAi 水稻株系米粉的DSC曲线。

Fig 2. Gelatinization properties of ALK-RNAi transgenic rice. A, Alkali spreading value of milled rice in 1.7% KOH; B, DSC curve of rice flours of ALK-RNAi transgenic rice.

表2 ALK-RNAi转基因水稻株系米粉的热力学特性和理化特性

Table 2 Gelatinization properties and physical-chemical qualities of ALK-RNAi transgenic rice.

转基因系	起始温度	峰值温度	终止温度	热焓值	直链淀粉含量	胶稠度	粗蛋白含量
Transgenic line	T_o /℃	T_p /℃	T_c /℃	△H /(J∙g^-1)	AAC/%	GC/mm	Protein content/%
龙特甫B LTFB	63.6±0.1	69.5±0.1	77.2±0.3	6.30±0.41	23.16±1.55	19.0±0.0		10.47±0.12
L-ALK^b-RNAi-1	61.8±0.4*	68.2±0.0**	76.2±0.5*	6.40±0.49	24.42±0.13	18.0±0.0		9.80±0.17
L-ALK^b-RNAi-2	62.2±0.3*	68.3±0.1**	76.0±0.5*	6.30±0.82	24.01±0.15	18.0±0.1		9.73±0.19
L-ALK^b-RNAi-3	62.2±0.5*	68.6±0.1**	76.0±0.0*	6.00±0.31	23.66±0.26	17.7±0.1		10.11±0.49
珍汕97B ZS97B	74.6±0.2	79.2±0.2	85.2±0.1	9.43±0.16	25.11±0.04	58.5±0.4		10.61±0.04
Z-ALK^c-RNAi-2-1	67.1±0.3**	73.4±0.1**	80.2±0.1**	8.20±0.08**	27.73±0.24**	25.5±0.1**		9.21±0.05*
Z-ALK^c-RNAi-2-2	66.3±0.0**	72.9±0.1**	79.4±0.2**	7.99±0.12**	27.85±0.13**	28.0±0.3**		9.65±0.42*
Z-ALK^c-RNAi-2-3	65.6±0.1**	72.4±0.1**	79.0±0.0**	7.29±0.07**	26.69±0.01**	45.0±0.0**		9.03±0.46*

图3 ALK-RNAi转基因稻米的RVA曲线(A)和淀粉聚合度分析(B)

Fig. 3. RVA curve (A) and the degree of polymerization (B) of rice flours from different ALK-RNAi lines.

表3 ALK-RNAi 转基因水稻株系米粉的RVA谱特征值

Table 3 RVA parameters of mature rice flour in ALK-RNAi transgenic rice lines.

转基因系 Transgenic line	峰值黏度 Peak viscosity /cP	热浆黏度 Hot paste viscosity/cP	崩解值Breakdown /cP	冷胶黏度 Cool paste viscosity /cP	消减值 Setback /cP	峰值时间 Peak time /min	起浆温度 Pasting temperature /℃
龙特甫B LTFB	3699±11	3217±18	482±7	6385±18	2686±5	6.5	87.9
L-ALK^b-RNAi-1	3923±7**	3390±10**	533±13**	6390±11	2467±8**	6.5	87.0*
L-ALK^b-RNAi-2	3810±13**	3312±25**	498±8*	6055±7**	2245±6**	6.5	86.9*
L-ALK^b-RNAi-3	3843±8**	3137±17*	706±10**	6345±14*	2502±7**	6.5	87.8*
珍汕97B ZS97B	2885±12	2510±16	375±9	4974±15	2089±9	6.5	88.6
Z-ALK^c-RNAi-2-1	3608±9**	2945±17**	663±12**	4749±17**	1141±11**	6.5	76.5**
Z-ALK^c-RNAi-2-2	3930±11**	3249±13**	681±11**	4590±16**	660±12**	6.6	77.4**
Z-ALK^c-RNAi-2-3	3976±6**	3337±14**	639±8**	4956±19	980±14**	6.5	76.5**

表4 ALK-RNAi转基因水稻株系稻米食味值

Table 4 Sensory evaluation of milled rice in ALK-RNAi transgenic rice lines.

样品 Sample	转基因系 Transgenic line	综合 Integrated score	外观 Appearance	口感 Taste
热饭 Cooked rice	龙特甫B LTFB	53.3±1.3	5.0±0.1	4.5±0.2
	L-ALK^b-RNAi-1	48.3±1.3**	4.3±0.1**	3.9±0.2*
	L-ALK^b-RNAi-2	51.0±0.8*	4.7±0.1*	4.2±0.1*
	L-ALK^b-RNAi-3	50.5±1.7**	4.6±0.2*	4.1±0.2*
	珍汕97B ZS97B	55.0±2.6	5.1±0.3	4.8±0.3
	Z-ALK^c-RNAi-2-1	43.8±1.9**	3.6±0.3**	3.3±0.3**
	Z-ALK^c-RNAi-2-2	42.0±0.8**	3.5±0.0**	3.1±0.1**
	Z-ALK^c-RNAi-2-3	44.5±0.6**	3.8±0.0**	3.4±0.0**
冷饭 Cooled rice	龙特甫B LTFB	49.8±0.5	4.6±0.0	3.9±0.1
	L-ALK^b-RNAi-1	44.8±0.5**	4.1±0.1**	3.6±0.1**
	L-ALK^b-RNAi-2	48.5±1.0*	4.3±0.1*	3.7±0.1*
	L-ALK^b-RNAi-3	46.5±0.6**	4.3±0.1*	3.7±0.1*
	珍汕97B ZS97B	54.0±0.8	5.1±0.0	4.7±0.1
	Z-ALK^c-RNAi-2-1	45.8±0.5**	3.0±1.4**	3.4±0.0**
	Z-ALK^c-RNAi-2-2	44.8±1.7**	3.4±0.2*	3.1±0.4**
	Z-ALK^c-RNAi-2-3	47.8±2.1**	4.0±0.2	3.6±0.3**

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