拟南芥ACOL8基因在乙烯合成与响应中的功能分析

doi:10.13560/j.cnki.biotech.bull.1985.2022-0560

摘要/Abstract

摘要：

植物激素乙烯在多种生理生化过程中发挥重要作用，但其在特定组织器官中的合成机制尚不完全清楚。拟南芥中存在12个功能未知的ACC氧化酶类似蛋白（ACO-like homolog，ACOL），运用基因定点编辑技术构建了ACOL8的功能丧失型突变体，发现该基因的突变削弱了经典的乙烯“三重反应”。与野生型相比，突变体黄化幼苗下胚轴及主根的长度显著增加，这与突变体对外源ACC的敏感性下降现象一致。同时还发现ACOL8基因的表达受乙烯信号的正反馈调控，EIN3过表达增强其表达水平，而etr1-3的突变则产生相反效应。再者，在正常条件下，ACOL8基因的突变并未影响拟南芥的生长；但在盐胁迫条件下，突变体的根冠比显著下降，这说明该基因参与植物的盐胁迫响应。综上，这些结果说明ACOL8可能具有ACC氧化酶的功能，参与乙烯的合成与响应。

关键词: ACC氧化酶类似蛋白, CRISPR/Cas9基因编辑技术, 乙烯合成, 耐盐性, 拟南芥

Abstract:

The plant hormone ethylene plays pivotal roles in various physiological and biochemical processes. However, the mechanisms controlling its synthesis in certain tissues and organs are not completely understood. There are 12 ACC oxidase homologs（ACO-like homolog, ACOL）of unknown functions in Arabidopsis thaliana. The loss-of-function mutants of ACOL8 gene were constructed using site-directed gene editing technology. It was found that the mutations of this gene attenuated the classical ethylene triple response. Compared with wild type, the mutants showed significant increases in the length of hypocotyls and primary roots of etiolated seedlings, which was consistent with the observation that the mutants had decreased sensitivity to exogenous ACC. Additionally, it was evident that the expression of this gene was subjected to positive feedback regulation of ethylene signaling, as exemplified by the finding that overexpression of EIN3 increased the expression of ACOL8 gene, whereas the etr1-3 muation exerted the opposite effect. Furthermore, the mutation of ACOL8 gene did not appear to affect the growth of A. thaliana under normal conditions, whereas the root-shoot ratio of the mutants decreased significantly under salt stress conditions, suggesting its involvement in plant salt stress response. Collectively, these results indicate that ACOL8 may function as an ACC oxidase to medaite ethylene synthesis and response.

Key words: ACC oxidase-like homolog, CRISPR/Cas9 gene editing technology, ethylene synthesis, salt tolerance, Arabidopsis thaliana

林蓉, 郑月萍, 徐雪珍, 李丹丹, 郑志富. 拟南芥ACOL8基因在乙烯合成与响应中的功能分析[J]. 生物技术通报, 2023, 39(1): 157-165.

LIN Rong, ZHENG Yue-ping, XU Xue-zhen, LI Dan-dan, ZHENG Zhi-fu. Functional Analysis of ACOL8 Gene in the Ethylene Synthesis and Response in Arabidopsis thaliana[J]. Biotechnology Bulletin, 2023, 39(1): 157-165.

图/表 13

图1 拟南芥ACOL8基因结构及基因编辑靶位点示意图

Fig. 1 Diagram of the structure of Arabidopsis ACOL8 gene and the target sites for gene editing

表1 用于ACOL8基因编辑的靶序列

Table 1 Target sequences for ACOL8 gene editing

靶基因 Target gene	sgRNA序列 sgRNA sequence（5'-3'）	位置 Location
ACOL8	TCTTTCGAGGAGACTATGACAGG	外显子（1）
ACOL8	GCACGTCGAGCGGGATCCCGTGG	外显子（1）

表2 引物序列

Table 2 Primer sequences

引物名称 Primer name	引物序列 Primer sequence（5'-3'）	用途 Utility
ZYP209-BsF	ATATATggtctcGATTGCTTTCGA- GGAGACTATGACGTT	构建sgRNA表达盒
ZYP210-F0	TGCTTTCGAGGAGACTATGAC- GTTTTAGAGCTAGAAATAGC	构建sgRNA表达盒
ZYP211-R0	AACCGGGATCCCGCTCGACGT- GCAATCTCTTAGTCGACTCTAC	构建sgRNA表达盒
ZYP212-BsR	ATTATTggtctcGAAACCGGGAT- CCCGCTCGACGTGC	构建sgRNA表达盒
ZYP237-FP	ATTCCTTTGATGCCGTGATAGT	鉴定筛选，测序
ZYP238-RP	TAGCTGGAACCTCTTTGATTCC	鉴定筛选
ZYP239-FP	ATCGATCTGAACGGAGGAGTAG	鉴定筛选
ZYP240-RP	TTTCGAGTGTGATCACGAGAGT	鉴定筛选，测序

表3 实时荧光定量PCR引物序列

Table 3 Sequences of the primers used in real-time fluore-scent quantitative PCR

引物名称 Primer name	基因 Gene	用途 Purpose	引物序列 Primer sequence（5'-3'）
ZZ200 qPCR-FP	ACTIN	内参基因	GTCGTACAACCGGTATTGTGCT
ZZ201 qPCR-RP	ACTIN	内参基因	TGTCTCTTACAATTTCCCGCTCT
ZYP197 qPCR-FP	ACOL8	目标基因	GGGGCTCTTGTCGTTAACCT
ZYP198 qPCR-RP	ACOL8	目标基因	TCCATATACTCGATGGCTCTCC

表4 编码拟南芥ACC氧化酶及其类似蛋白的基因特性

Table 4 Characteristics of genes encoding Arabidopsis ACC oxidases and ACO-like homologs

基因 Gene	基因ID Gene ID	编码蛋白 Encoded protein	氨基酸 Amino acid/aa
ACO1	AT2G19590	ACO1	310
ACO2	AT1G62380	ACO2	320
ACO3	AT1G12010	ACO3	320
ACO4	AT1G05010	ACO4	323
ACO5	AT1G77330	ACO5	307
ACOL1	AT1G06620	ACO-like homolog 1	365
ACOL2	AT1G06640	ACO-like homolog 2	369
ACOL3	AT1G06650	ACO-like homolog 3	369
ACOL4	AT1G03400	ACO-like homolog 4	351
ACOL5	AT1G03410	ACO-like homolog 5	398
ACOL6	AT1G04350	ACO-like homolog 6	360
ACOL7	AT1G04380	ACO-like homolog 7	345
ACOL8	AT3G61400	ACO-like homolog 8	370
ACOL9	AT5G43440	ACO-like homolog 9	365
ACOL10	AT5G43450	ACO-like homolog 10	362
ACOL11	AT5G59530	ACO-like homolog 11	364
ACOL12	AT5G59540	ACO-like homolog 12	366

图2 ACC氧化酶家族及其类似蛋白的系统树图分析 MEGA7根据氨基酸序列比对的结果将序列分群，归为同一群的蛋白具有比较亲近的关系

Fig. 2 A dendrogram analysis of members of the ACC oxidase family and ACO-like homologs The MEGA7 software groups the sequences according to the results of amino acid sequence alignment，and the proteins classified into the same group have a relatively close relationship

图3 ACC氧化酶家族及ACOL8的氨基酸序列比对相同颜色的氨基酸位点具有同源性，黑色=100%，红色>75%，绿色>50%；sgRNA指基因编辑位点对应的氨基酸序列

Fig. 3 Amino acid sequences alignment of the ACC oxidase family members and ACOL8 Amino acid sites with the same color indicate homology，black=100%，red>75%，green>50%；sgRNA refers to the amino acid sequence corresponding to the gene editing site

表5 三个acol8突变体的突变位点

Table 5 Mutation sites in the three acol8 mutants

株系Line	突变体编号Mutant code	突变类型Mutation type	突变位点Mutation site	突变特点Mutation characteristics
acol8-1	A10/WT-14-5-38	插入	308-309：5 bp插入CTGGA	移码突变
acol8-2	A10/WT-230-17	缺失	66-308：243 bp缺失	大片段缺失
acol8-3	A10/WT-254-19	插入	65-66：1 bp插入T 308-309：1 bp插入T	提前终止

图4 三个acol8突变体的突变序列

Fig. 4 Mutated sequences in the three acol8 mutants

图5 acol8突变体与野生型拟南芥黄化幼苗的“三重反应”比较 A：acol8突变体与野生型拟南芥黄化幼苗在不同浓度ACC处理下的表型，标尺= 2 mm；B：acol8突变体与野生型拟南芥黄化幼苗在不同浓度ACC处理下的下胚轴长；C：acol8突变体与野生型拟南芥黄化幼苗在不同浓度ACC处理下的主根长。不同字母表示存在0.05水平上的显著性差异，下同

Fig. 5 Comparison of the “triple responses” of etiolated seedlings of wild type Arabidopsis and acol8 mutant A：The phenotype of etiolated seedlings of acol8 single mutant and wild type Arabidopsis treated with different concentrations of ACC，Bar = 2 mm. B：Length of hypocotyl of etiolated seedlings of acol8 single mutants and wild-type Arabidopsis treated with different concentrations of ACC. C：Length of primary root of etiolated seedlings of acol8 single mutants and wild type Arabidopsis. Different letters indicate significant differences at the 0.05 level，the same below

图6 etr1-3和 EIN3ox与野生型拟南芥的ACOL8基因相对表达量比较

Fig. 6 Comparison of the relative expressions of ACOL8 gene in the roots of wild type Arabidopsis and etr1-3 and EIN3ox line

图7 三个acol8突变体与野生型拟南芥的表型比较 A：acol8突变体与野生型拟南芥的地上部表型，标尺=1 cm；B：acol8突变体与野生型拟南芥的地上部鲜重。

Fig. 7 Comparison of the phenotypes of wild type Arabid-opsis and three acol8 mutants A：The aboveground phenotype of wild type Arabidopsis and acol8 single mutant，Bar=1 cm. B：The fresh weight of aboveground biomass of wild type Arabidopsis and acol8 single mutants

图8 acol8突变体与野生型拟南芥的耐盐性比较 acol8突变体与野生型拟南芥在不同浓度NaCl处理下的根冠比（A）和表型比较（B），标尺=1 cm

Fig. 8 Comparison of salt tolerances of wild type Arabido-psis and acol8 mutants Comparison of the root-shoot ratio （A）and the phenotype （B）of wild type Arabi-dopsis with that of acol8 mutant under different concentrations of NaCl，Bar=1 cm

参考文献 20

[1]	Ahammed GJ, Gantait S, Mitra M, et al. Role of ethylene crosstalk in seed germination and early seedling development:a review[J]. Plant Physiol Biochem, 2020, 151:124-131. doi: 10.1016/j.plaphy.2020.03.016 URL
[2]	Nascimento VL, Pereira AM, Pereira AS, et al. Physiological and metabolic bases of increased growth in the tomato ethylene-insensitive mutant Never ripe:extending ethylene signaling functions[J]. Plant Cell Rep, 2021, 40(8): 1377-1393. doi: 10.1007/s00299-020-02623-y pmid: 33074436
[3]	Tripathi SK, Tuteja N. Integrated signaling in flower senescence[J]. Plant Signal Behav, 2007, 2(6): 437-445. doi: 10.4161/psb.2.6.4991 URL
[4]	Dubois M, van den Broeck L, Inzé D. The pivotal role of ethylene in plant growth[J]. Trends Plant Sci, 2018, 23(4): 311-323. doi: S1360-1385(18)30015-3 pmid: 29428350
[5]	Tao JJ, Chen HW, Ma B, et al. The role of ethylene in plants under Sal Inity stress[J]. Front Plant Sci, 2015, 6:1059.
[6]	Kazan K. Diverse roles of jasmonates and ethylene in abiotic stress tolerance[J]. Trends Plant Sci, 2015, 20(4): 219-229. doi: 10.1016/j.tplants.2015.02.001 pmid: 25731753
[7]	Yu YB, Adams DO, Yang SF. 1-Aminocyclopropanecarboxylate synthase, a key enzyme in ethylene biosynthesis[J]. Arch Biochem Biophys, 1979, 198(1): 280-286. pmid: 507845
[8]	Adams DO, Yang SF. Ethylene biosynthesis:identification of 1-aminocyclopropane-1-carboxylic acid as an intermediate in the conversion of methionine to ethylene[J]. Proc Natl Acad Sci USA, 1979, 76(1): 170-174. doi: 10.1073/pnas.76.1.170 pmid: 16592605
[9]	Park CH, Roh J, Youn JH, et al. Arabidopsis ACC oxidase 1 coordinated by multiple signals mediates ethylene biosynthesis and is involved in root development[J]. Mol Cells, 2018, 41(10): 923-932.
[10]	Ning Q, Jian YN, Du YF, et al. An ethylene biosynthesis enzyme controls quantitative variation in maize ear length and kernel yield[J]. Nat Commun, 2021, 12(1): 5832. doi: 10.1038/s41467-021-26123-z pmid: 34611160
[11]	Chersicola M, Kladnik A, Žnidarič MT, et al. 1-aminocyclopropane-1-carboxylate oxidase induction in tomato flower pedicel phloem and abscission related processes are differentially sensitive to ethylene[J]. Front Plant Sci, 2017, 8:464. doi: 10.3389/fpls.2017.00464 pmid: 28408916
[12]	Penarrubia L, Aguilar M, Margossian L, et al. An antisense gene stimulates ethylene hormone production during tomato fruit ripening[J]. Plant Cell, 1992, 4(6): 681-687. doi: 10.2307/3869526 URL
[13]	Lincoln JE, Fischer RL. Diverse mechanisms for the regulation of ethylene-inducible gene expression[J]. Mol Gen Genet, 1988, 212(1): 71-75. doi: 10.1007/BF00322446 URL
[14]	Wang ZP, Xing HL, Dong L, et al. Egg cell-specific promoter-controlled CRISPR/Cas9 efficiently generates homozygous mutants for multiple target genes in Arabidopsis in a single generation[J]. Genome Biol, 2015, 16(1): 144. doi: 10.1186/s13059-015-0715-0 URL
[15]	朱丽颖, 郑月萍, 徐雪珍, 等. 一种准确、简便测定CRISPR/Cas9基因编辑效率的方法[J]. 江苏农业学报, 2020, 36(2): 299-305.
	Zhu LY, Zheng YP, Xu XZ, et al. A convenient and accurate method for determining the efficiency of CRISPR/Cas9-based gene editing[J]. Jiangsu J Agric Sci, 2020, 36(2): 299-305.
[16]	Giovannoni JJ. Genetic regulation of fruit development and ripening[J]. Plant Cell, 2004, 16(Suppl):S170-S180.
[17]	Yin XR, Allan AC, Chen KS, et al. Kiwifruit EIL and ERF genes involved in regulating fruit ripening[J]. Plant Physiol, 2010, 153(3): 1280-1292. doi: 10.1104/pp.110.157081 URL
[18]	Riyazuddin R, Verma R, Singh K, et al. Ethylene:a master regulator of Sal Inity stress tolerance in plants[J]. Biomolecules, 2020, 10(6): 959. doi: 10.3390/biom10060959 URL
[19]	Jiang CF, Belfield EJ, Cao Y, et al. An Arabidopsis soil-Sal Inity-tolerance mutation confers ethylene-mediated enhancement of sodium/potassium homeostasis[J]. Plant Cell, 2013, 25(9): 3535-3552. doi: 10.1105/tpc.113.115659 URL
[20]	Lockhart J. Salt of the earth:ethylene promotes salt tolerance by enhancing Na/K homeostasis[J]. Plant Cell, 2013, 25(9): 3150. doi: 10.1105/tpc.113.250911 URL