垂枝与直枝梅花PmTAC1启动子序列差异与垂枝性状的关系初探

doi:10.16420/j.issn.0513-353x.2021-0468

摘要/Abstract

摘要：

在前期转录组数据的基础上对梅花（Prunus mume）垂枝候选基因PmTAC1（TILLER ANGLE CONTROL 1）进行了克隆及相关分析。PmTAC1编码区全长915 bp,编码304个氨基酸,且在梅花垂枝和直枝品种中无序列差异。氨基酸序列比对进一步证实PmTAC1具备IGT家族典型结构域,进化树分析表明PmTAC1与桃（Prunus persica）的PpeTAC1亲缘关系最近,亚细胞定位预测该蛋白定位于细胞核和叶绿体,蛋白疏水性分析显示其具有亲水性。PmTAC1在茎中表达最高,叶和顶芽次之。PmTAC1在垂枝品种的1年生枝条中的表达显著高于直枝品种,且在两种枝型枝条近远轴侧均有差异。垂枝品种PmTAC1的启动子序列长度为1 608 bp,直枝品种启动子序列长度为1 379 bp。启动子顺式元件预测到光响应、赤霉素合成、脱落酸响应顺式元件的数量在两种枝型品种间具有差异。推测PmTAC1在垂枝和直枝梅花品种中的启动子序列及表达量差异可能与垂枝性状形成有关。

关键词: 梅花, 垂枝性状, PmTAC1, 基因克隆, 表达

Abstract:

A candidate gene PmTAC1（TILLER ANGLE CONTROL 1）of the weeping trait of Prunus mume was cloned and analyzed based on our previous transcriptome data. The coding sequence of PmTAC1 was 915 bp,encoding 304 amino acids,and no differences was observed between upright and weeping cultivars of mei. The amino acid sequence alignment further confirmed that PmTAC1 had the typical domain of the IGT family,and evolutionary analysis showed that the genetic relationship between PmTAC1 and PpeTAC1 was the closest. Prediction of subcellular localization and hydrophobic analysis of protein showed that the protein was located in the nucleus and chloroplast,and was hydrophilic. The expression of PmTAC1 in branches of weeping cultivars was significantly higher than that in 1-year-old branches of upright cultivars,and there were differences in the adaxial and abaxial of the two types of branches. The promoter sequences of PmTAC1 in weeping and upright mei were 1 608 bp and 1 379 bp respectively,which were consistent among different cultivars of the same branch types of mei. Differences were detected in the number of light responses,gibberellin,and abscisic acid response cis-elements between the two promoter sequences. In conclusion,the promoter sequence and expression level of PmTAC1 in weeping and upright cultivars of mei may be related to the formation of weeping traits.

Key words: Prunus mume, weeping trait, PmTAC1, gene cloning, expression

中图分类号:

S685.17

刘瑶瑶, 吴严严, 石岩, 毛天宇, 包满珠, 张俊卫, 张杰. 垂枝与直枝梅花PmTAC1启动子序列差异与垂枝性状的关系初探[J]. 园艺学报, 2022, 49(6): 1327-1338.

LIU Yaoyao, WU Yanyan, Shi Yan, MAO Tianyu, BAO Manzhu, ZHANG Junwei, ZHANG Jie. Preliminary Study on the Relationship Between Promoter Sequence Difference of PmTAC1 and Weeping Trait of Prunus mume[J]. Acta Horticulturae Sinica, 2022, 49(6): 1327-1338.

图/表 11

图1 垂枝与直枝梅花品种表型 C1：磨山垂枝;C2：粉红垂枝;C3：淡粉垂枝;Z1：米单绿;Z2：小绿萼;Z3：单瓣朱砂。下同。

Fig. 1 Phenotypes of mei cultivars with weeping and upright branches C1：Moshan Chuizhi;C2：Fenhong Chuizhi;C3：Danfen Chuizhi;Z1：Midanlü;Z2：Xiaolü E;Z3：Danban Zhusha. The same below.

图2 直枝枝条（左）与垂枝枝条（右）取样示意图（Mao et al.,2020）

Fig. 2 Schematic diagram of sampling of upright（leaf）and weeping（right）branches of Mei（Mao et al.,2020）

表1 PmTAC1克隆和表达量分析所用引物

Table 1 Primers used for cloning and expression analyses of PmTAC1

用途 Primer use	引物名称 Primer name	引物序列（5′-3′） Primer sequnce
克隆Cloning	PmTAC1-F	ATGAAGATCTTCAACTGGGTTC
	PmTAC1-R	TCAGTGCACACAAGGGGCACCTTG
启动子克隆Promoter cloning	PmTAC1-Pro-F	TGAAGCCTCTTATGAAC
	PmTAC1-Pro-R	CAGAAAGTAACCCACCT
实时荧光定量qRT-PCR	PmTAC1-qRTF	AAACCAATGACAAGGACACAC
	PmTAC1-qRTR	CGCTCTCCAGAACGAAAT
	ACTIN2- qRTF	TGTGATGGTTGGTATGGGG
	ACTIN2- qRTR	AACTGGATGCTCTTCTGGG

图3 梅花PmTAC1编码区的克隆

Fig. 3 Cloning of coding sequences of PmTAC1 of Mei M：Marker DL5000.

图4 PmTAC1及其他物种TAC1氨基酸序列的系统发育树

Fig. 4 Phylogenetic tree of PmTAC1 amino acid sequences of Mei and other species

图5 梅花PmTAC1在垂枝品种C2和直枝品种Z3不同组织中的表达 t-检验。* α = 0.05,** α= 0.01。

Fig. 5 Expression of PmTAC1 in different tissues of weeping cultivar C2 and upright cultivar Z3 t-test. *α = 0.05,**α = 0.01.

图6 梅花PmTAC1在F1群体垂枝和直枝后代枝条近轴和远轴侧中的表达不同字母代表差异显著（P < 0.05）。

Fig. 6 Expression of PmTAC1 in the adaxial and abaxial sides of the branches from the weeping and upright offspring of F1 population Different letters mean significant difference（P < 0.05）. T-test*a = 0.05,**a = 0.01.

图7 梅花PmTAC1在垂枝品种C1、C2、C3和直枝品种Z1、Z2、C3枝条近轴和远轴侧中的表达不同字母代表差异显著（P < 0.05）。

Fig. 7 Expression of PmTAC1 in adaxial and abaxial sides of weeping cultivar C1,C2,C3 and upright cultivar Z1,Z2,Z3 Different letters mean significant difference（P < 0.05）.

图8 梅花PmTAC1启动子克隆片段的琼脂糖凝胶电泳

Fig. 8 Agarose gel electrophoresis of amplified fragment of PmTAC1 promoter of Mei M：DNA marker DL5000.

图9 梅花垂枝和直枝中PmTAC1启动子碱基序列比对

Fig. 9 Base sequence alignment of PmTAC1 promoter in weeping and upright Mei

表2 梅垂枝和直枝PmTAC1启动子顺式元件分布及功能注释

Table 2 Promoter cis-element distribution and functional annotations of PmTAC1

cis-Element	垂枝Weeping	直枝 Upright	功能 Function
GATA-motif	1	1	光响应Part of a light responsive element
I-box	2	2	光响应Part of a light responsive element
AAAC-motif	1	0	光响应Light responsive element
GT1-motif	2	0	光响应Light responsive element
G-box	0	1	光响应cis-Acting regulatory element involved in light responsiveness
TCCC-motif	0	1	光响应Part of a light responsive element
CAAT-box	29	24	增强子Common cis-acting element in promoter and enhancer regions
ARE	2	1	厌氧诱导cis-Acting regulatory element essential for the anaerobic induction
circadian	1	1	昼夜节律控制cis-Acting regulatory element involved in circadian control
TC-rich repeats	1	1	防御和应激cis-Acting element involved in defense and stress responsiveness
LTR	1	1	低温响应cis-Acting element involved in low-temperature responsiveness
O2-site	1	1	玉米醇溶酶蛋白代谢调节cis-Acting regulatory element involved in zein metabolism regulation
TATA-box	51	29	核心启动子Core promoter element around -30 of transcription start
TCA-element	1	1	水杨酸响应cis-Acting element involved in salicylic acid responsiveness
ABRE	0	1	脱落酸响应cis-Acting element involved in the abscisic acid responsiveness
P-box	0	1	赤霉素响应Gibberellin-responsive element
CGTCA-motif	1	1	茉莉酸甲酯响应cis-Acting regulatory element involved in the MeJA-responsiveness
TGACG-motif	1	1	茉莉酸甲酯响应cis-Acting regulatory element involved in the MeJA-responsiveness

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