Grain Quality as Affected by Down-regulation of Expression of Different ALK Alleles in indica Rice (Oryza sativa L.)

doi:10.16819/j.1001-7216.2019. 9090

Abstract

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

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

CLC Number:

Q755
S511.01

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.

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.

Figures/Tables 7

References 34

[1]	陈静. 江苏省水稻食味改良育种研究进展. 江苏农业科学, 2015, 43(12): 77-80.
	Chen J.A review of the research on the improvement of rice taste in Jiangsu Province.Jiangsu Agric Sci, 2015, 43(12): 77-80. (in Chinese)
[2]	Tian Z X, Qian Q, Liu Q Q, Yan M X, Liu X F, Yan C J, Liu G F, Gao Z Y, Tang S Z, Zeng D L, Wang Y H, Yu J M, Gu M H, Li J Y.Allelic diversities in rice starch biosynthesis lead to diverse array of rice eating and cooking qualities.Proc Natl Acad Sci USA, 2009, 106(51): 21 760-21 765.
[3]	Xu C W, Mo H D.Qualitative-quantitative analysis for inheritance of gelatinization temperature in indica rice(O. sativa subsp. indica). Acta Agron Sin, 1996, 22(4): 386-389.
[4]	Khush G S, Paule C M, De La Cruz, N M. Rice grain quality evaluation and improvement at IRRI//The Workshop on Chemical Aspects of Rice Grain Quality, 1978.
[5]	Chun A, Lee H J, Hamaker B R, Janaswamy S.Effects of ripening temperature on starch structure and gelatinization, pasting, and cooking properties in rice (Oryza sativa). J Agric Food Chem, 2015, 63(12): 3085-3093.
[6]	Nakamura Y, Francisco J P, Hosaka Y, Sato A, Sawada T, Kubo A, Fujita N.Essential amino acids of starch synthase IIa differentiate amylopectin structure and starch quality between japonica and indica rice varieties. Plant Mol Biol, 2005, 58(2): 213-227.
[7]	Umemoto T, Yano M, Satoh H, Shomura A, Nakamura Y.Mapping of a gene responsible for the difference in amylopectin structure betweenjaponica-type and indica-type rice varieties. Theor Appl Genet, 2002, 104(1): 1-8.
[8]	Gao Z Y, Zeng D L, Cui X, Zhou Y H, Yan M X, Huang D N, Li J Y, Qian Q.Map-based cloning of theALK gene, which controls the gelatinization temperature of rice. Sci China C Life Sci, 2003, 46(6): 661-668.
[9]	Bao J S, Sun M, Zhu L H, Corke H.Analysis of quantitative trait loci for some starch properties of rice (Oryza sativa L.): Thermal properties, gel texture and swelling volume. J Cereal Sci, 2004, 39(3): 379-385.
[10]	Fan C C, Yu X Q, Xing Y Z, Xu C G, Luo L J, Zhang Q F.The main effects, epistatic effects and environmental interactions of QTLs on the cooking and eating quality of rice in a doubled-haploid line population.Theor Appl Genet, 2005, 110(8): 1445-1452.
[11]	Ebadi A A, Farshadfar E, Rabiei B.Mapping QTLs controlling cooking and eating quality indicators of Iranian rice using RILs across three years.Aust J Crop Sci, 2013, 7(10): 1494-1502.
[12]	Zhao W G, Chung J W, Kwon S W, Lee J H, Ma K H, Park Y J.Association analysis of physicochemical traits on eating quality in rice (Oryza sativa L.). Euphytica, 2013, 191(1): 9-21.
[13]	Gao Z Y, Zeng D L, Cheng F M, Tian Z X, Guo L B, Su Y, Yan M X, Jiang H, Dong G J, Huang Y C, Han B, Li J Y, Qian Q.ALK, the key gene for gelatinization temperature,is a modifier gene for gel consistency in rice. J Integr Plant Biol, 2011, 53(9): 756-765.
[14]	Nakamura Y, Francisco P Y, Sato A, Sawada T, Kubo A, Fujita N.Essential amino acids of starch synthase IIa differentiate amylopectin structure and starch quality betweenjaponica and indica rice varieties. Plant Mol Biol, 2005, 58(2): 213-227.
[15]	Cuevas R P, Daygon V D, Corpuz H, Nora L, Reinke R, Waters D, Fitzgerald M.Melting the secrets of gelatinisation temperature in rice.Funct Plant Biol, 2010, 37(5): 439-447.
[16]	Bao J S, Peng X, Hiratsuka M, Sun M, Umemoto T.Granule-bound SSIIa protein content and its relationship with amylopectin structure and gelatinization temperature of rice starch.Starch-Stärke, 2010, 61(8): 431-437.
[17]	Kharabianmasouleh A, Waters D L E, Reinke R F, Ward R, Henry R J. SNP in starch biosynthesis genes associated with nutritional and functional properties of rice.Sci Rep, 2012, 2: 557.
[18]	Waters D L E, Henry R J, Reinke R F, Fitzgerald M A. Gelatinization temperature of rice explained by polymorphisms in starch synthase.Plant Biotechnol J, 2010, 4(1): 115-122.
[19]	Chen M H, Bergman C J, Fjellstrom R G.SSSIIA locus genetic variation associated with alkali spreading value in international rice germplasm. Proceedings of the Plant and Animal Genome Conference, 2003, 153.
[20]	Bao J S, Corke H, Sun M.Nucleotide diversity in starch synthase IIa and validation of single nucleotide polymorphisms in relation to starch gelatinization temperature and other physicochemical properties in rice (Oryza sativa L.). Theor Appl Genet, 2006, 113(7): 1171-1183.
[21]	Zhou Y, Zheng H Y, Wei G C, Zhou H, Han Y N, Bai X F, Xing Y Z, Han Y P.Nucleotide Diversity and Molecular Evolution of the ALK Gene in Cultivated Rice and its Wild Relatives. Plant Mol Biol Rep, 2016, 34(5): 1-8.
[22]	陈秀花, 刘巧泉, 王宗阳, 王兴稳, 蔡秀玲, 张景六, 顾铭洪. 反义Wx基因导入我国籼型杂交稻重点亲本. 科学通报, 2002, 47(9): 684-688.
	Chen X H, Liu Q Q, Wang Z Y, Wang X W, Cai X L, Zhang J L, Gu M H.Introduction of antisense Wx Gene into key parents of indica hybrid rice in China. Chin Sci Bull, 2002, 47(9): 684-688. (in Chinese)
[23]	Murray M G, Thompson W F.Rapid isolation of high molecular weight plant DNA.Nucl Acids Res, 1980, 8(19): 4321-4325.
[24]	Zhang C Q, Zhou L H, Zhu Z B, Lu H W, Zhou X Z, Qian Y T, Li Q F, Lu Y, Gu M H, Liu Q Q.Characterization of grain quality and starch fine structure of two japonica rice(Oryza sativa) cultivars with good sensory properties at different temperatures during the filling stage. J Agric Food Chem, 2016, 64(20): 4048-4057.
[25]	Zhu L J, Meng X L, Shi Y C, Gu M H, Liu Q Q.High-amylose rice improves indices of animal health in normal and diabetic rats.Plant Biotechnol J, 2012, 10(3): 353-362.
[26]	Umemoto T, Horibata T, Aoki N, Hiratsuka M, Yano M, Inouchi N.Effects of variations in starch synthase on starch properties and eating quality of rice.Plant Prod Sci, 2008, 11(4): 472-480.
[27]	Miura S, Crofts N, Saito Y, Hosaka Y, Oitome N F, Watanabe T, Kumamaru T, Fujita N.Starch Synthase IIa-deficient mutant rice line produces endosperm starch with lower gelatinization temperature than japonica rice cultivars. Front Plant Sci, 2018, 9: 1-10.
[28]	Umemoto T, Aoki N, Lin H, Nakamura Y, Inouchi N, Sato Y.Natural variation in rice starch synthase IIa affects enzyme and starch properties.Funct Plant Biol, 2004, 31(7): 671-684.
[29]	Kaw R N, Cruz N M D L. Genetic analysis of amylose content, gelatinization temperature and gel consistency in rice.J Genet Breed, 1990: 103-111.
[30]	李晓方, 肖昕, 刘彦卓, 刘志霞, 卢东柏, 毛兴学, 杨俊, 方正武, 李志新. 籼稻稻米品质性状遗传特点新解析. 分子植物育种, 2009, 7(6): 1077-1083.
	Li X F, Xiao X, Liu Y Z, Liu Z X, Lu D B, Mao X X, Yang J, Fang Z W, Li Z X.Novel anlysis on genetic characters of quality traits in indica rice.Mol Plant Breed, 2009, 7(6): 1077-1083. (in Chinese with English abstract)
[31]	隋炯明, 李欣, 严松, 严长杰, 张蓉, 汤述翥, 陆驹飞, 陈宗祥, 顾铭洪. 稻米淀粉RVA谱特征与品质性状相关性研究. 中国农业科学, 2005, 38(4): 657-663.
	Sui J M, Li X, Yan S, Yan C J, Zhang R, Tang S Z, Lu J F, Chen Z X, Gu M H.Studies on the rice RVA profile characteristics and its correlation with the quality.Sci Agric Sin, 2005, 38(4): 657-663. (in Chinese with English abstract)
[32]	曹清明, 钟海雁, 李忠海, 孙昌波. 蕨根淀粉糊化温度测定及影响因素研究. 食品与机械, 2007, 23(3): 16-19.
	Cao Q M, Zhong H Y, Li Z H, Sun C B.Studies on the rice RVA profile characteristics and its correlation with the quality. Food & Mach, 2007, 23(3): 16-19. (in Chinese with English abstract)
[33]	张昌泉, 潘立旭, 周兴忠, 李钱峰, 刘巧泉. 不同Wx等位组合对杂交稻天优3611品质的影响 . 中国科技论文在线, 2007.
	Zhang C Q, Pan L X, Zhou X Z, Li Q F, Liu Q Q.The combined effects of different Wx allele on grain quality of hybrid rice Tianyou 3611. Chin Sci Paper,2007.
[34]	刘鑫燕, 朱孔志, 张昌泉, 洪燃, 孙鹏, 汤述翥, 顾铭洪, 刘巧泉. 利用9311来源的粳型染色体片段代换系定位控制稻米糊化温度的微效QTL. 作物学报, 2014, 40(10): 1740-1747.
	Liu X Y, Zhu K Z, Zhang C Q, Hong R, Sun P, Tang S Z, Gu M H, Liu Q Q.Mapping of minor QTLs for rice gelatinization temperature using chromosome segment substitution lines from indica 9311 in the japonica background. Acta Agron Sin, 2014, 40(10): 1740-1747. (in Chinese with English abstract)

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

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

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*

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*

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**