BnABCI8影响甘蓝型油菜叶绿体发育

doi:10.3724/SP.J.1006.2023.24065

摘要/Abstract

摘要：

成熟叶绿体是高等植物光合作用的重要场所, 是影响作物产量的重要器官。BnABCI8是ABC转运蛋白I亚族的成员, 在甘蓝型油菜中有2个功能拷贝, 分别是BnA09.ABCI8和BnC09.ABCI8, 其氨基酸序列在不同物种中是非常保守的。表达模式分析发现, BnABCI8在油菜植株各个组织中均有表达, 且在叶和花中表达量较高; 亚细胞定位证明, BnABCI8能够定位在叶绿体中; 表型鉴定发现, BnA09.ABCI8和BnC09.ABCI8的同时突变及BnA09.ABCI8的单突变均会导致黄色的子叶和褪绿的真叶, 且双突变体褪绿更为严重; 透射电镜结果显示, 双突变体中叶绿体不能够形成正常的类囊体膜; BnABCI8的敲除导致叶绿素合成途径相关基因的表达下调, 且叶片中积累了大量的Fe离子。这些结果表明, BnABCI8的突变造成叶绿体结构异常, 叶绿素合成受阻, 叶片中Fe离子大量积累, 而Fe离子的积累又可能会引发一系列的反应如活性氧积累, 细胞死亡和叶绿素降解等, 最终导致了叶色突变。

关键词: 甘蓝型油菜, BnABCI8, ABC转运蛋白, 叶绿体发育, 叶绿素合成, 杂色突变体

Abstract:

Mature chloroplast is an important place for photosynthesis of higher plants and important organs that affects crop yield. BnABCI8 is a member of the ABC transporter I subfamily, and there are two functional copies of BnA09.ABCI8 and BnC09.ABCI8 in Brassica napus. Their amino acid sequences are very conserved in different species. The relative expression patterns showed that BnABCI8 was expressed in all tissues of Brassica napus, and the relative expression level in leaves and flowers was higher. Subcellular localization indicated that BnABCI8 was located in chloroplast. Phenotypic identification showed that the double mutation of BnA09.ABCI8 and BnC09.ABCI8 and the single mutation of BnA09.ABCI8 both resulted in yellow cotyledons and chlorotic true leaves, among which double mutant was more severe chlorosis. Transmission electron microscope demonstrated that the chloroplasts in the double mutants could not form normal thylakoid membranes. The knock-out of BnABCI8 resulted in the decrease of the relative expression level of related genes in the chlorophyll synthesis pathway, and significantly increased the iron content in mutant leaves. These results indicated that the mutation of BnABCI8 resulted in abnormal chloroplast structure, hindered the synthesis of chlorophyll, and significantly increased the iron content in the leaves. In addition, the accumulation of iron ion might lead to a series of reactions such as accumulation of reactive oxygen species, cell death and chlorophyll degradation, and eventually led to mutation of leaf color.

Key words: Brassica napus, BnABCI8 gene, ABC transporter, chloroplast development, chlorophyll synthesis, variegated mutant

陈晓汉, 王丽琴, 汪华栋, 肖清, 陶保龙, 赵伦, 文静, 易斌, 涂金星, 傅廷栋, 沈金雄. BnABCI8影响甘蓝型油菜叶绿体发育[J]. 作物学报, 2023, 49(4): 893-905.

CHEN Xiao-Han, WANG Li-Qin, WANG Hua-Dong, XIAO Qing, TAO Bao-Long, ZHAO Lun, WEN Jing, YI Bin, TU Jin-Xing, FU Ting-Dong, SHEN Jin-Xiong. BnABCI8 affects chloroplast development of Brassica napus[J]. Acta Agronomica Sinica, 2023, 49(4): 893-905.

图/表 12

附表1

本研究中用到的引物"

引物名称 Primer name	引物序列 Primer sequence (5'-3')	作用 Function
C83-A09F	cttgcatgcctgcaggtcgacATGGCGTCTCTCCTCGCG	亚细胞定位 Subcellular localization assay
C83-A09R	tctaccggtacccggggatccGAACCCACTGATCCTTCAAGCTT
C83-C09FF	cttgcatgcctgcaggtcgacATGGCGTCTCTCCTCGCG
C83-C09RR	tctaccggtacccggggatccGAACCCACTGATCCTTCAAGCTT
C83F	ATTTCATTTGGAGAGGACCTCG
C83R	TGTGCCCATTAACATCACCATC
QA09F	ATCCAGGTTAAGAATCCATCAGCG	qPCR 分析 qPCR analysis
QA09R	TCTGCAGAACCCTGAGATCATCG
QC09F	ATCCAGGTTAAGAACCCATCAGCC
QC09R	TCTGCAGAACCCAGAGATCATTG
QHEMA1F	TCCGAGGAACAACAACAGAACCAG
QHEMA1R	TGTATATCTGTCGGGTGCAGAGT
QHEMCF	CGGTTCCGTCTCCGTCAT
QHEMCR	GGCATTAGGTGCGGATTCAG
QHEME2F	CGAAATCTTCCCGGTCAATTCGTTG
QHEME2R	AGAGGCTCAGTGGTTGCAGAG
QCHLHF	GCACGCTTGGTTTGCATCCTATT
QCHLHR	CATGTGACTTCCCTGTTCTAGGGTCA
QCHLDF	GATTGCTATTTCAGGTCGTAGAGG
引物名称 Primer name	引物序列 Primer sequence (5'-3')	作用 Function
QCHLDR	GTTCGTCTTCCCACTCATCAG
QCHLMF	GCGACGATCGTTTCCTTGAC
QCHLMR	AAATACTCCCTCACCACCTCCTT
QDVRF	TCCAGTGGATACATACAACCAATGGCT
QDVRR	ATTGGTTTGGAAGGTGGAAGCGAGA
QPORCF	CCGACAAGATCTCCATCAAGGAG
QPORCR	CGGCGATGCTTCGTTCGAT
QCHLGF	CGAGTTGGAGCACTCTCTCTCCA
QCHLGR	CTCTTCCCAGAAGAGTTGGAGTCC
QCAOF	GTCTATTGTCTTCCTTCTTCCTC
QCAOR	TCCTTTCACTCCCTTCTTTCTG
actionQ3F	CTATCCTCCGTCTCGATCTCGC
actionQ3R	CTTAGCCGTCTCCAGCTCTTGC
HA09-1-F	ggagtgagtacggtgtgcACAGTACTTCCAAAACCTAGACTACGAC	Hi-tom 分析 Hi-tom analysis
HA09-1-R	gagttggatgctggatggGCCGAGCTTGTCGAAATACTCGA
HA09-2-F	ggagtgagtacggtgtgcCCGCTTTGAACTCAGCTGTGT
HA09-2-R	gagttggatgctggatggCTTCGTGACGAAATTATAAATCCCTCCT
HC09-1-F	ggagtgagtacggtgtgcGCAATACTTCCAAAACCTAGACTACGAC
HC09-1-R	gagttggatgctggatggGCCGAGCTTGTCGAAGTAGTCAA
HC09-2-F	ggagtgagtacggtgtgcCTGCTCTGAACTCCGCCG
HC09-2-R	gagttggatgctggatggGTGACGAAATTGTAGATCCCTCCCTTC
F-1	ACTCTTTCCCTACACGACGCTCTTCCGATCTgcttGCGTtggagtgagtacggtgtgc
F-2	ACTCTTTCCCTACACGACGCTCTTCCGATCTgcttGTAGtggagtgagtacggtgtgc
F-3	ACTCTTTCCCTACACGACGCTCTTCCGATCTgcttACGCtggagtgagtacggtgtgc
F-4	ACTCTTTCCCTACACGACGCTCTTCCGATCTgcttCTCGtggagtgagtacggtgtgc
F-5	ACTCTTTCCCTACACGACGCTCTTCCGATCTgcttGCTCtggagtgagtacggtgtgc
F-6	ACTCTTTCCCTACACGACGCTCTTCCGATCTgcttAGTCtggagtgagtacggtgtgc
F-7	ACTCTTTCCCTACACGACGCTCTTCCGATCTgcttCGACtggagtgagtacggtgtgc
F-8	ACTCTTTCCCTACACGACGCTCTTCCGATCTgcttGATGtggagtgagtacggtgtgc
F-9	ACTCTTTCCCTACACGACGCTCTTCCGATCTgcttATACtggagtgagtacggtgtgc
F-10	ACTCTTTCCCTACACGACGCTCTTCCGATCTgcttCACAtggagtgagtacggtgtgc
F-11	ACTCTTTCCCTACACGACGCTCTTCCGATCTgcttGTGCtggagtgagtacggtgtgc
F-12	ACTCTTTCCCTACACGACGCTCTTCCGATCTgcttACTAtggagtgagtacggtgtgc
R-A	GACTGGAGTTCAGACGTGTGCTCTTCCGATCTctgtGCGTtgagttggatgctggatgg
R-B	GACTGGAGTTCAGACGTGTGCTCTTCCGATCTctgtGTAGtgagttggatgctggatgg
R-C	GACTGGAGTTCAGACGTGTGCTCTTCCGATCTctgtACGCtgagttggatgctggatgg
R-D	GACTGGAGTTCAGACGTGTGCTCTTCCGATCTctgtCTCGtgagttggatgctggatgg
R-E	GACTGGAGTTCAGACGTGTGCTCTTCCGATCTctgtGCTCtgagttggatgctggatgg
R-F	GACTGGAGTTCAGACGTGTGCTCTTCCGATCTctgtAGTCtgagttggatgctggatgg
R-G	GACTGGAGTTCAGACGTGTGCTCTTCCGATCTctgtCGACtgagttggatgctggatgg
R-H	GACTGGAGTTCAGACGTGTGCTCTTCCGATCTctgtGATGtgagttggatgctggatgg
5UDI427	AATGATACGGCGACCACCGAGATCTACACGCCTTCAAACACTCTTTCCCTACACGACGC
5UDI428	AATGATACGGCGACCACCGAGATCTACACAGGAACCTACACTCTTTCCCTACACGACGC
7UDI427	CAAGCAGAAGACGGCATACGAGATTCACGAAGGTGACTGGAGTTCAGACGTGTGCTCTT
引物名称 Primer name	引物序列 Primer sequence (5'-3')	作用 Function
7UDI428	CAAGCAGAAGACGGCATACGAGATCTTAGCCAGTGACTGGAGTTCAGACGTGTGCTCTT
Cas9F	CGAGAAGAAGAACGGCCTGTTCG	基因编辑 Gene editing
Cas9R	AGTTGCCCCTAGCGAGTGGG
U626-IDF	TGTCCCAGGATTAGAATGATTAGGC
U629-IDR	AGCCCTCTTCTTTCGATCCATCAAC
crp1-BsF-ABCI8	ATATATGGTCTCGATTGACTCCTTCACAATCCCCAAGTT
crp1-F0-ABCI8	TGACTCCTTCACAATCCCCAAGTTTTAGAGCTAGAAATAGC
crp2-R0-ABCI8	AACCGTGTTCTTGGGGATATAGCAATCTCTTAGTCGACTCTAC
crp2-BsR-ABCI8	ATTATTGGTCTCGAAACCGTGTTCTTGGGGATATAGCAA

附表1

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