水稻褐色颖壳新等位基因OsFBX310bh6的克隆鉴定

doi:10．3969/j．issn．1001G7216．2015．04．001

摘要/Abstract

摘要：

通过EMS诱变获得遗传稳定的水稻褐色颖壳突变体(brown hull 6, 简称bh6)。与野生型IR64相比,在自然条件下突变体的颖壳颜色自抽穗期开始逐渐加深直至蜡熟期颖壳呈深褐色,除千粒重和穗长显著降低外,其他农艺性状(株高、每株有效穗数、每穗实粒数、结实率)未发生显著改变。遗传分析表明,该褐色颖壳性状受一对隐性核基因控制。构建突变体bh6与正常色颖壳水稻CPSLO17的F₂分离群体作为基因定位群体,将该基因精细定位在第9染色体长臂上106 kb的区段内。序列分析发现,突变体中F-box蛋白基因OsFBX310 (LOC_Os09g12150)编码区第1013位碱基由G突变为A,致使其所编码的蛋白序列第338位的甘氨酸(Gly)突变成天冬氨酸(Asp)。通过构建OsFBX310功能互补载体进行转化试验,结果表明,突变体转基因植株的谷壳颜色恢复为正常颜色。该基因是已报道的水稻OsFBX310基因的新等位基因,命名为OsFBX310^bh6。OsFBX310^bh6及其野生型等位基因的克隆对深入研究338位甘氨酸/天冬氨酸在类黄酮代谢过程中的作用,以及丰富水稻特异种质资源具有积极意义。

关键词: 水稻, 褐色颖壳突变体, 分子标记, OsFBX310, 类黄酮代谢

Abstract:

A brown hull 6 (bh6) mutant of rice was isolated from an EMS-induced IR64 mutant bank. The brown pigments start to accumulate in the hulls after heading and reach a maximum level in mature seeds. The 1000-grain weight and panicle length of bh6 significantly decreased while other agronomic traits including plant height, number of productive panicles per plant, number of filled grains per panicle and seed-setting rate were largely similar to those of the wild-type IR64. Analysis of pigment contents indicated that the levels of total flavonoids and anthocyanin in bh6 were significantly increased. The brown hull phenotype was controlled by a single recessive gene on the long arm of chromosome 9. Sequencing analysis indicated that a single base substitution (G-A) at position 1013 was detected in the candidate gene (LOC_Os09g12150) encoding an F-box domain containing protein. Functional complementation using the wild-type allele could rescue the mutation. The mutation, designated as OsFBX310^bh6, is a new allele of OsFBX310 functioning as an inhibitor of brown hull. Isolation of OsFBX310^bh6 provides useful experimental materials and would facilitate the studies on mechanism of flavonoid metabolism in monocot plants.

Key words: Oryza sativa L., brown hull mutant, molecular marker, OsFBX310, flavonoid metabolism

中图分类号:

Q343.5
Q785

徐霞, 张晓波, 施勇烽, 王惠梅, 黄奇娜, 奉保华, 李小红, 郭丹, 吴建利. 水稻褐色颖壳新等位基因OsFBX310^bh6的克隆鉴定[J]. 中国水稻科学, 2015, 29(4): 335-342.

Xia XU, Xiao-bo ZHANG, Yong-feng SHI, Hui-mei WANG, Qi-na HUANG, Bao-hua FENG, Xiao-hong LI, Dan GUO, Jian-li WU. Identification of OsFBX310^bh6, A New Allele for Brown Hull in Rice[J]. Chinese Journal OF Rice Science, 2015, 29(4): 335-342.

图/表 8

图1 野生型IR64与突变体bh6的表型

Fig.1. Phenotype of IR64 and the mutant bh6.

表1 基因定位所用的分子标记

Table 1 Molecular markers used for gene mapping.

标记 Marker	正向引物 Forward primer (5'-3')	反向引物 Reverse primer (5'-3')
RM23804	GAGCCGACTCAATCCAATCCTCTCC	ATCGTCTTCCAAGCAAGTGCAAGC
RM23893	GGCGGCTTAAGAGTGTTTGTAGG	AGAGGATTGCTTTGCTGATGAGG
RM23904	CTCACCGGAGCACCACTAACC	GAGAGCAAGACTGTGAAGTGTGAACC
RM7038	GATTAGAGCTTTGGTGGTTCTTGG	ACTTGTGGTCGGTCTGGTAGTCC
InDel-6	GAATCCGGTTCCGAGACTAAA	TCAATCTTGCAGCAAACAAGGC
InDel-10	ACCTGATCGATGTCTAATTTGCAC	CGTTTACGAAAGGCAGAGGACA

表2 突变体和野生型的农艺性状比较(平均值±标准差)

Table 2 Comparison of agronomic traits between wild type IR64 and the mutant bh6(Mean±SD).

材料 Material	株高 Plant height /cm	穗长 Panicle length /cm	每株有效穗数 No. of productive panicles per plant	每穗实粒数 No. of filled grains per panicle	结实率 Seed-setting rate/%	千粒重 1000-grain weight/g
IR64	120.0±1.5	27.4±0.3	15±3	89±12	71.6±1.3	24.3±0.06
bh6	119.4±1.4	26.1±0.4^*	16±2	81±16	70.7±1.6	22.5±0.13^**

图2 颖壳中总黄酮和花青素的含量

Fig. 2. Contents of total flavonoid and anthocyanin in rice hulls.

表3 褐色颖壳突变体bh6 的遗传分析

Table 3 Genetic analysis of the brown hull mutant bh6.

组合 Cross	F₁	F₂			P_(3∶1)
组合 Cross	F₁	总植株数 Total no. of plants		野生型表型单株数 No. of wild type plants	突变表型单株数 No. of mutant type plants
bh6/CPSLO17	正常颜色颖壳Normal	2107	1600	507	0.32
bh6/IR64	正常颜色颖壳Normal	684	507	177	0.60

图3 突变体基因在第9染色体上的精细定位(A-突变座位bh6与SSR标记RM23804、RM23893、RM23904和RM7038连锁; B-利用877个F2隐性单株将bh6定位在Indel-6与Indel-10约106 kb的区间; C-基因编码区第1013碱基处,碱基G突变为A。)

Fig. 3. Fine mapping of the mutation on chromosome 9. (A, bh6 is linked with SSR markers RM23804, RM23893, RM23904 and RM7038; B, bh6 is located in a 106 kb region between InDel-6 and InDel-10 based on 877 F2 plants; C, Single base substitution at position 1013 (G1013A).)

表4 bh6/IR64的3个F3分离株系的遗传分析

Table 4 Genetic analysis of F3 segregation lines derived from bh6/ IR64.

F₃株系 F₃ line	总植株数 Total no. of plants	野生型表型单株数 No. of wild type plants	突变表型单株数 No. of mutant type plants	P_(3:1)
株系1 Line 1	276	216	60	0.21
株系2 Line 2	212	163	49	0.53
株系3 Line 3	132	107	25	0.11

图4 功能互补验证(A-互补载体; B-野生型、突变体与互补植株的表型; C-互补植株突变位点序列分析。箭头表示突变位点。)

Fig. 4. Functional complementation of the mutation. (A, Complementation construct; B, Phenotype of IR64, bh6 and complementary transgenic line; C, Sequencing analysis of complementary transgenic lines. The arrow refers to the mutation site.)

参考文献 29

[1]	Quattrocchio F, Verweij W, Kroon A, et al.PH4 of petunia is an R2R3MYB protein that activates vacuolar acidification through interactions with basic-helix-loophelix transcription factors of the anthocyanin pathway.Plant Cell, 2006, 18: 1274-1291.
[2]	Nagao S.Genetic analysis and linkage relationship of characters in rice.Adv Genet, 1951, 4: 181-212.
[3]	Takahashi M.Analysis of aciculus color genes essential to anthocyanin coloration in rice.J Fac Agric Hokkaido Univ, 1957, 50: 266-362.
[4]	Reddy A R.Genetic and molecular analysis of the anthocyanin pigmentation pathway in rice//Khush G S. Rice Genetics: Ⅲ. Proceedings of the Third International Rice Genetics Symposium, 16-20, Oct 1995. Manila, Philippines: International Rice Research Institute, 1996: 341-352.
[5]	Kinoshita T.Current linkage maps.Rice Genet Newsl, 1984, 1: 16-27.
[6]	Saitoh K, Onishi K, Mikami I, et al.Allelic diversification at the C (OsC1) locus of wild and cultivated rice: Nucleotide changes associated with phenotypes.Genetics, 2004, 168: 997-1007.
[7]	Nagao S, Takahashi M.Trail construction of twelve linkage groups in Japanese rice: ⅩⅩⅦ. Genetical studies on rice plant.J Fac Agric Hokkaido Univ, 1963, 53: 72-130.
[8]	Iwata N, Omura T.Linkage analysis by reciprocal translocation method in rice plants (Oryza Sativa L.):Ⅱ. Linkage groups corresponding to the chromosomes 5, 6, 8, 9, 10 and 11.Sci Bull Fac Agric Kyushu Univ, 1971, 25: 137-153.
[9]	Iwata N, Omura T.Linkage studies in rice plants (Oryza sativa L.) on some mutants derived from chronic gamma irradiation.J Fac Agric Kyushu Univ, 1977, 21: 117-127.
[10]	李亮杰, 周海鹏, 占小登, 等. 一个水稻金黄色颖壳和节间基因的遗传定位. 中国水稻科学, 2008, 22(4): 432-434.
[11]	李秋圆, 叶胜海, 张迎迎, 等. 一个水稻金黄色颖壳与节间基因的定位. 核农学报, 2012, 26(7): 983-987.
[12]	Cui J J, Fan S C, Shao T, et al.Characterization and fine mapping of the ibf mutant in rice.J Integr Plant Biol, 2007, 49(5): 678-685.
[13]	Maekawa M.Geographical distribution of the genes for black hull coloration.Rice Genet Newsl, 1984, 1: 104-105.
[14]	Gu X Y, Kianian S F, Hareland G A, et al.Genetic analysis of adaptive syndromes interrelated with seed dormancy in weedy rice (Oryza sativa).Theor Appl Genet, 2005, 110: 1108-1118.
[15]	Zhu B F, Si L, Wang Z, et al.Genetic control of a transition from black to straw-white seed hull in rice domestication.Plant Physiol, 2011, 155: 1301-1311.
[16]	Hong L L, Qian Q, Tang D, et al.A mutation in the rice chalcone isomerase gene causes the golden hull and internode 1 phenotype.Planta, 2012, 236: 141-151.
[17]	Zhang K W, Qian Q, Huang Z J, et al.Gold hull and inter-node2 encodes a primarily multifunctional cinnamyl-alcohol dehydrogenase in rice.Plant Physiol, 2006, 140(3): 972-983.
[18]	Mirecki R M, Teramura A H.Effects of ultraviolet-B irradiation on soybean: V. The dependence of plants sensitivity on the photosynthetic photon flux density during and after leaf expansion.Plant Physiol, 1984, 74: 475-480.
[19]	Shao T, Qian Q, Tang D, et al.A novel gene IBF1 is required for the inhibition of brown pigment deposition in rice hull furrows.Theor Appl Genet, 2012, 125: 381-390.
[20]	卢扬江, 郑康乐. 提取水稻DNA 的一种简易方法. 中国水稻科学, 1992, 6(1): 47-48.
[21]	朱正歌, 肖晗, 傅亚萍, 等. 水稻转座子突变体库的构建及突变类型的遗传分析. 生物工程学报, 2001, 17: 288-292.
[22]	Lepiniec L, Debeaujon I, Routaboul J M, et al.Genetics and biochemistry of seed flavonoids.Annu Rev Plant Biol, 2006, 57: 405-430.
[23]	Shirely B W.Flavonoid biosynthesis: “New” functions for an“old” pathway.Trends Plant Sci, 1996, 1: 377-382.
[24]	Pourecl L, Rouraboul J M, Kerhoas L, et al.TRANSPARENT TESTA10 encodes a laccase-like enzyme involved in oxidative polymerization of flavonoids in Arabidopsis seed coat.Plant Cell, 2005, 17: 2966-2980.
[25]	韩磊, 吴先军, 张红军, 等. 黑米果皮花色素沉积过程的研究. 中国水稻科学, 2006, 20(4): 384-388.
[26]	Reddy A R, Scheffler B, Madhuri G, et al.Genetic and molecular analysis of the anthocyanin pigmentation pathway in rice.Plant Mol Biol, 1996, 32: 735-743.
[27]	Druka A, Kudrna D, Rostoks N, et al.Chalcone isomerase gene from rice(Oryza sativa) and barley (Hordeum vulgare): Physical, genetic and mutation mapping.Gene, 2003, 302: 171-178.
[28]	Jain M, Nijhawan A, Arora R, et al.F-Box proteins in rice: Genome-wide analysis, classification, temporal and spatial gene expression during panicle and seed development, and regulation by light and abiotic stress.Plant Physiol, 2007, 143: 1467-1483.
[29]	Reddy V S, Dash S, Reddy A R.Anthocyanin pathway in rice (Oryza sativa L.): Identification of a mutant showing dominantinhibition of anthocyanins in leaf and accumulation of proantho-cyanidins in pericarp.Theor Appl Genet, 1995, 91(2): 301-312.