水稻淡绿叶突变体HM133的遗传分析与基因定位

doi:10.16819/j.1001-7216.2016.6033

摘要/Abstract

摘要：

EMS诱导籼稻品种IR64获得淡绿叶突变体HM133。与野生型IR64相比,HM133播种后的第6周和第15周的光合色素含量以及抽穗期的净光合速率显著降低,气孔导度则明显上升;此外,突变体株高、每穗实粒数和结实率等农艺性状也较野生型显著下降。叶绿体超微结构分析表明,分蘖期HM133类囊体基粒片层形状不规则,堆叠凌乱、排列疏松。遗传分析表明HM133淡绿叶性状受单隐性核基因控制。通过分子标记将该基因定位于第3染色体长臂RM143和RM3684之间。该区间内包含编码镁螯合酶D亚基的基因OsCHLD。序列分析表明HM133中该基因第10外显子上有一个从G突变为A的单碱基变异,导致编码的氨基酸由精氨酸变成谷氨酸,推测OsCHLD基因即为控制HM133淡绿叶表型的候选基因。

关键词: 水稻, 淡绿叶, 基因定位, 镁螯合酶, 光合作用

Abstract:

The pale green leaf mutant HM133 was identified from an EMS-induced IR64 mutant bank. The contents of photosynthetic pigments including chlorophyll and carotenoid of HM133 were reduced significantly at 6 weeks and 15 weeks after sowing when compared with IR64.The net photosynthetic rate of HM133 was considerably lower than that of the wild-type IR64 at heading stage while the stomatal conductance was apparently increased. The agronomic traits including plant height, number of filled grain per panicle and seed-setting rate decreased significantly in the mutant compared with the wild-type. In addition, the mutant exhibited a less number of grana, irregular arrangement of thylakoid layer in the chloroplast at the tillering stage. Genetic and mapping analysis showed that the pale green phenotype was controlled by a single recessive gene located in the long arm of chromosome 3 between SSR markers RM143 and RM3684. The interval contains an ORF OsChlD encoding magnesium-chelatase D subunit. Sequence analysis revealed that the mutant allele carried a nucleotide substitution from G to A in the tenth exon of OsChlD, which led to the substitution of glutamic acid for arginine acid. Therefore, it is deduced that OsChlD is the candidate gene controlling the pale green leaf phenotype of HM133.

Key words: Oryza sativa, pale green leaf, gene mapping, magnesium-chelatase, photosynthesis

中图分类号:

施勇烽, 贺彦, 郭丹, 吕向光, 黄奇娜, 吴建利. 水稻淡绿叶突变体HM133的遗传分析与基因定位[J]. 中国水稻科学, 2016, 30(6): 603-610.

Yong-feng SHI, Yan HE, Dan GUO, Xiang-guang LV, Qi-na HUANG, Jian-li WU. Genetic Analysis and Gene Mapping of a Pale Green Leaf Mutant HM133 in Rice[J]. Chinese Journal OF Rice Science, 2016, 30(6): 603-610.

图/表 8

表1 实时定量PCR引物

Table 1 Primers used in real-time PCR.

基因 Gene	正向引物(5'-3') Forward primer(5'-3')	反向引物(5'-3') Reverse primer(5'-3')
OsChlD	GGAAAGAGAGGGCATTAG	CAATACGATCAAGTAAGTGTT
OsChlI	AGTAACCTTGGTGCTGTG	AATCCATCAACATTCAACTCTG
OsChlH	CTATACATTCGCCACACT	TATCACACAACTCCCAAG
HEMA1	CGCTATTTCTGATGCTATGGGT	TCTTGGGTGATGATTGTTTGG
PORA	TGTACTGGAGCTGGAACAACAA	GAGCACAGCAAAATCCTAGACG
CAO1	GATCCATACCCGATCGACAT	CGAGAGACATCCGGTAGAGC
Cab1R	AGATGGGTTTAGTGCGACGAG	TTTGGGATCGAGGGAGTATTT
Cab2R	TGTTCTCCATGTTCGGCTTCT	GCTACGGTCCCCACTTCACT
psaA	GCGAGCAAATAAAACACCTTTC	GTACCAGCTTAACGTGGGGAG
psbA	CCCTCATTAGCAGATTCGTTTT	ATGATTGTATTCCAGGCAGAGC
rbcL	CTTGGCAGCATTCCGAGTAA	ACAACGGGCTCGATGTGATA
rbcS	TCCGCTGAGTTTTGGCTATTT	GGACTTGAGCCCTGGAAGG
YGL1	CAGTCTCCAATGGCCACCT	TGCTTTCATCAGTGGCTGG
SPP	CGGAGAGGAAACATAATGAC	ATAGGCATTTGTCTTTGTCTC
PPR1	CTAAGACCGAATGACAAATGC	GCACTGCCAACAAGAATACC
DVR	CGAGCCCAGGTTCATCAAGGTGC	CCTCCCGATCTTGCCGAACTCC
Ubiquitin	GCTCCGTGGCGGTATCAT	CGGCAGTTGACAGCCCTAG

图1 野生型IR64和突变体HM133的不同时期植株表型 A—野生型IR64和突变体HM133幼苗表型;B—野生型IR64和突变体HM133分蘖期的表型。

Fig. 1. Phenotype of the wide-type IR64 and the mutant HM133 at different growth stages. A, Phenotype of the wide type IR64 and the mutant HM133 at the seedling stage; B, Phenotype of the wide type IR64 and the mutant HM133 at the tillering stage.

表2 野生型IR64和突变体HM133的主要农艺性状

Table 2 Agronomic traits of the wild-type IR64 and mutant HM133.

材料 Material	株高 Plant height /cm	有效穗数 No. of productive panicles	穗长 Panicle length /cm	每穗实粒数 Number of filled grains per panicle	结实率 Seed-setting rate/%	千粒重 1000-grain weight/g
IR64	113.0±1.7	14.0±1.0	25.0±1.4	81.2±7.1	74.6±1.6	27.52±0.38
HM133	108.7±0.6^*	12.7±2.1	24.9±0.3	62.9±7.3^*	68.8±2.1^*	28.35±0.35

表3 不同生长时期HM133与IR64叶片光合色素含量的比较

Table 3 Comparison of photosynthetic pigment contents between HM133 and IR64 at different growth stages. mg/g

取样时间 Sampling time	材料 Material	叶绿素a Chlorophyll a content	叶绿素b Chlorophyll b content	总叶绿素 Total chlorophyll content	类胡萝卜素 Carotenoid content
播种后6周 6 weeks after sowing	IR64	3.76±0.57	1.00±0.14	4.78±0.67	0.85±0.19
	HM133	2.11±0.06^**	0.46±0.04^**	2.58±0.03^**	0.52±0.10^*
播种后15周 15 weeks after sowing	IR64	2.86±0.29	0.88±0.13	3.77±0.42	0.63±0.04
	HM133	1.54±0.08^**	0.42±0.03^**	1.97±0.12^**	0.33±0.01^**

表4 抽穗期野生型IR64和突变体HM133的剑叶光合特性

Table 4 Photosynthetic parameters of flag leaf of IR64 and HM133 at the heading stage.

材料 Material	净光合速率 P_n/(μmol·m^-2s^-1)	气孔导度 G_S/(mol·m^-2s^-1)	胞间CO₂浓度 C_i/(μmol·mol^-1)	蒸腾速率 T_r/(mmol·m^-2s^-1)
IR64	14.45±1.78	0.57±0.13	309.13±7.30	4.12±0.64
HM133	12.74±1.38^*	0.73±0.09^*	313.75±6.41	4.26±0.25

图2 野生型IR64和突变体HM133的叶绿体超微结构 A和B-野生型IR64; C和D-突变体HM133。 S-淀粉粒; C-叶绿体; G-基粒; SL-基质片层; OG-嗜饿颗粒。

Fig. 2. Chloroplast ultrastructure of IR64 and HM133. A and B, IR64; C and D, HM133. S, Starch granule; C, Chloroplast; G, Granum; SL, Stroma lamella; OG, Osmiophilic granule.

图3 pglHM133定位及候选基因预测 A-pglHM133初步定位; B-候选基因OsChlD序列分析. 灰色部分表示HM133的1541位碱基G突变为A; C-IR24/HM133 F2群体的Taq Ⅰ酶切验证. M-分子量标记; 1-IR24; 2-HM133; 3-F1; 4~9-F2中淡绿叶单株; 10~25-F2群体中正常叶单株。

Fig. 3. Location of pglHM133 and candidate gene prediction. A,Primary mapping of yglHM133 on chromosome 3; B, Sequence analysis of the candidate gene OsChlD. The gray letters indicate single base substitution from G to A at position 1541; C, Mutation base of OsChlD confirmed with Taq I restriction enzyme digestion. M, Molecular marker; 1, IR24; 2, HM133; 3, F1; 4-9, Pale green plant of F2; 10-25, Normal plant of F2.

图4 RT-PCR分析野生型IR64与HM133 中叶绿素合成、叶绿体发育相关基因的表达

Fig. 4. Expression of genes associated with chlorophyll biosynthesis and chloroplast development in IR64 and HM133 by real-time PCR.

参考文献 29

[1]	李智强, 朱丹, 王志龙, 等. 水稻黄绿叶突变体djyg的遗传分析与基因定位. 中国水稻科学, 2015, 29: 601-609.
	Li Z Q, Zhu D, Wang Z L,et al.Genetic analysis and gene mapping of a yellow-green leaf mutant djyg in rice. Chin J Rice Sci, 2015, 29: 601-609. (in Chinese with English abstract)
[2]	Jung K H, Hur J, Ryu C H, et al.Characterization of a rice chlorophyll-deficient mutant using the T-DNA gene-trap system.Plant Cell Physiol, 2003, 44: 463-472.
[3]	韩帅, 王立静, 钟世宜, 等. 一个新的玉米叶色突变体的遗传分析及基因定位. 玉米科学, 2012, 20: 26-28.
	Han S, Wang L J,Zhong S Y, et al.Genetic analysis and gene mapping of a new leaf color mutant in maize.J Maize Sci, 2012, 20: 26-28. (in Chinese with English abstract)
[4]	Sawers R J, Viney J, Farmer P R, et al.The maize Oil yellow1 (Oy1) gene encodes the I subunit of magnesium chelatase. Plant Mol Biol, 2006, 60: 95-106.
[5]	Axelsson E, Lundqvist J, Sawicki A, et al.Recessiveness and dominance in barley mutants deficient in Mg-chelatase subunit D, an AAA protein involved in chlorophyll biosynthesis.Plant Cell, 2006, 18: 3606-3616.
[6]	Mochizuki N,Brusslan J A, Larkin R, et al. Arabidopsis genomes uncoupled 5 (GUN5) mutant reveals the involvement of Mg-chelatase H subunit in plastid-to-nucleus signal transduction. Proc Natl Acad Sci, 2001, 98: 2053-2058.
[7]	肖华贵, 杨焕文, 饶勇, 等. 甘蓝型油菜黄化突变体的叶绿体超微结构、气孔特征参数及光合特性. 中国农业科学, 2013, 46: 715-727.
	Xiao H G, Yang H W, Rao Y,et al.Analysis of chloroplast ultrastructure, stomatal characteristic parameters and photosynthetic characteristics of chlorophyll-reduced mutant in Brassica napus L. Sci Agric Sin, 2013, 46: 715-727. (in Chinese with English abstract)
[8]	Beale S I.Green genes gleaned.Trends Plant Sci, 2005, 10: 309-312.
[9]	Lee S, Kim J H, Yoo E S, et al.Differential regulation of chlorophyll a oxygenase genes in rice.Plant Mol Biol, 2005, 57: 805-818.
[10]	Zhang H, Li J, Yoo J H, et al.Rice Chlorina-1 and Chlorina-9 encode ChlD and ChlI subunits of Mg-chelatase, a key enzyme for chlorophyll synthesis and chloroplast development. Plant Mol Biol, 2006, 62: 325-337.
[11]	Wu Z, Zhang X, He B, et al.A chlorophyll-deficient rice mutant with impaired chlorophyllide esterification in chlorophyll biosynthesis.Plant Physiol, 2007, 145: 29-40.
[12]	Wang P, Gao J, Wan C, et al.Divinyl chlorophyll(ide) a can be converted to monovinyl chlorophyll(ide) a by a divinyl reductase in rice.Plant Physiol, 2010, 153: 994-1003.
[13]	Deng X J, Zhang H Q, Wang Y, et al.Mapped clone and functional analysis of leaf-color gene Ygl7 in a rice hybrid(Oryza sativa L. ssp. indica). PLoS One, 2014, 9(6): e99564.
[14]	Sakuraba Y, Rahman M L, Cho S H, et al.The rice faded green leaf locus encodes protochlorophyllide oxidoreductase B and is essential for chlorophyll synthesis under high light conditions.Plant J, 2013, 74: 122-133.
[15]	Li Q Z, Zhu F Y, Gao X, et al.Young Leaf Chlorosis 2 encodes the stroma-localized heme oxygenase 2 which is required for normal tetrapyrrole biosynthesis in rice.Planta, 2014, 240: 701-712.
[16]	Chen H, Cheng Z, Ma X, et al.A knockdown mutation of YELLOW-GREEN LEAF2 blocks chlorophyll biosynthesis in rice. Plant Cell Rep, 2013, 32: 1855-1867.
[17]	Li J, Pandeya D, Nath K, et al. ZEBRA-NECROSIS, a thylakoid-bound protein, is critical for the photoprotection of developing chloroplasts during early leaf development. Plant J, 2010, 62: 713-725.
[18]	Su N, Hu M L, Wu D X, et al.Disruption of a rice pentatricopeptide repeat protein causes a seedling-specific albino phenotype and its utilization to enhance seed purity in hybrid rice production.Plant Physiol, 2012, 159: 227-238.
[19]	Lv X G, Shi Y F, Xu X, et al. Oryza sativa chloroplast signal recognition particle 43 (OscpSRP43) is required for chloroplast development and photosynthesis. PLoS ONE, 2015, 10(11): e143249.
[20]	Zhang F, Luo X, Hu B, et al. YGL138(t), encoding a putative signal recognition particle 54 kDa protein, is involved in chloroplast development of rice. Rice, 2013, 6(1): 7.
[21]	Wellburn A R.The spectral determination of chlorophylls a and b, as well as total carotenoids, using various solvents with spectrophotometers of different resolution.J Plant Physiol, 1994, 144: 307-313.
[22]	Arnon D I.Copper enzymes in isolated chloroplasts. Polyphenoloxidase in Beta vulgaris. Plant Physiol, 1949, 24: 1-15.
[23]	Huang Q N, Shi Y F, Zhang X B, et al.Single base substitution in OsCDC48 is responsible for premature senescence and death phenotype in rice. J Integr Plant Biol, 2016, 58: 12-28.
[24]	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.
	Lu Y J, Zheng K L.A simple method for isolation of rice DNA.Chin J Rice Sci, 1992, 6(1): 47-48.(in Chinese with English abstract)
[25]	Shi Y F, Chen J, Liu W Q, et al.Genetic analysis and gene mapping of a new rolled-leaf mutant in rice (Oryza sativa L.). Sci China C Life Sci, 2009, 52: 885-890.
[26]	Masuda T.Recent overview of the Mg branch of the tetrapyrrole biosynthesis leading to chlorophylls.Photosynth Res, 2008, 96: 121-143.
[27]	孙小秋, 王兵, 肖云华, 等. 水稻ygl98黄绿叶突变基因的精细定位与遗传分析. 作物学报, 2011, 37(6): 991-997.
	Sun X Q, Wang B, Xiao Y H,et al.Genetic analysis and fine-mapping of ygl98 yellow-green leaf gene in rice. Acta Agron Sin, 2011, 37(6): 991-997. (in Chinese with English abstract).
[28]	Tian X Q, Ling Y H, Fang L K, et al.Gene cloning and functional analysis of yellow green leaf3 (ygl3) gene during the whole-plant growth stage in rice. Genes & Genom, 2013, 35: 87-93.
[29]	Luo T, Luo S, Araujo W L, et al.Virus-induced gene silencing of pea CHLI and CHLD affects tetrapyrrole biosynthesis, chloroplast development and the primary metabolic network.Plant Physiol Biochem, 2013, 65: 17-26.