园艺学报 ›› 2021, Vol. 48 ›› Issue (3): 505-517.doi: 10.16420/j.issn.0513-353x.2020-0520
收稿日期:
2020-07-08
出版日期:
2021-03-25
发布日期:
2021-04-02
通讯作者:
曹家树
E-mail:jshcao@ziu.edu.cn
基金资助:
Received:
2020-07-08
Online:
2021-03-25
Published:
2021-04-02
Contact:
CAO Jiashu
E-mail:jshcao@ziu.edu.cn
摘要:
在从白菜‘矮脚黄’自交系Bcajh97-01中克隆获得多聚半乳糖醛酸酶基因BcPG17及其启动子序列的基础上,采用qRT-PCR、原位杂交和启动子融合表达载体瞬时转化技术,分析了该基因的表达特征。BcPG17的DNA全长为2 105 bp,包含9个外显子和8个内含子。ORF序列长度为1 344 bp,编码447个氨基酸残基。预测的编码蛋白的相对分子量为48.61 kD,理论等电点为8.39,含有1个跨膜结构域,N末端具有信号肽序列,具有被分泌到细胞膜外起作用的特征;其氨基酸序列具有PG蛋白的4个典型结构域,与十字花科芸薹属的其他物种亲缘关系较近。通过qRT-PCR分析发现,BcPG17在开花期花茎中的表达水平最高,且在花粉发育的四分体时期和单核小孢子时期有表达。原位杂交试验结果也显示,BcPG17在开花期花茎的所有组织中均存在强烈的表达信号。通过对克隆获得的1 442 bp的BcPG17启动子序列分析,发现该序列含有与分生组织表达有关的顺式作用元件1个、与植物激素响应相关的作用基序和元件多个,以及花药特异基序4个和绒毡层降解延迟蛋白(TDR)结合位点2个;能够启动GUS信号在开花期花茎的第2节间和节处强烈表达,在花发育中期的花药中也存在明显的GUS信号。上述结果表明,BcPG17可能通过与植物激素代谢相关蛋白相互作用调控花茎的伸长,受到绒毡层降解相关蛋白的调控参与花粉的发育。
中图分类号:
吕美玲, 曹家树. 白菜花茎发育相关基因BcPG17的表达特征分析[J]. 园艺学报, 2021, 48(3): 505-517.
LÜ Meiling, CAO Jiashu. Expression Characteristic Analysis of the BcPG17,a Gene Related to Inflorescence Stem Development of Brassica campestris[J]. Acta Horticulturae Sinica, 2021, 48(3): 505-517.
物种(基因名称) Species(gene name) | GenBank登录号 GenBank No. | |||
---|---|---|---|---|
拟南芥Arabidopsis thaliana(PGA3) | NP_187439.1 | |||
拟南芥Arabidopsis thaliana(PGA2) | CAA51692.1 | |||
白菜Brassica campestris(BcMF2) | ABW24665.1 | |||
白菜Brassica campestris(BcMF6) | ACP74159.1 | |||
白菜Brassica campestris(BcMF9) | ABN13878.1 | |||
白菜Brassica campestris(BcMF16) | ADJ68232.1 | |||
油菜Brassica napus(RDPG) | CAA65072.1 | |||
油菜Brassica napus(PGAZ) | CAC05658.1 | |||
甜瓜Cucumis melo(MPG2) | AAC26511.1 | |||
陆地棉Gossypium hirsutum(G9) | AAA82167.1 | |||
荔枝Litchi chinensisv(LcPG1) | AFW04075.1 | |||
番茄Solanum lycopersicum(TAPG1) | AAC28903.1 | |||
番茄Solanum lycopersicum(TAPG2) | AAC28904.1 | |||
烟草Nicotiana tabacum(PG1) | Q05967.1 | |||
桃Prunus persica(PRF5) | CAA54150.1 |
表1 多序列比对分析所用的PG基因信息
Table 1 Details of the PG genes used in the multiple alignment analysis
物种(基因名称) Species(gene name) | GenBank登录号 GenBank No. | |||
---|---|---|---|---|
拟南芥Arabidopsis thaliana(PGA3) | NP_187439.1 | |||
拟南芥Arabidopsis thaliana(PGA2) | CAA51692.1 | |||
白菜Brassica campestris(BcMF2) | ABW24665.1 | |||
白菜Brassica campestris(BcMF6) | ACP74159.1 | |||
白菜Brassica campestris(BcMF9) | ABN13878.1 | |||
白菜Brassica campestris(BcMF16) | ADJ68232.1 | |||
油菜Brassica napus(RDPG) | CAA65072.1 | |||
油菜Brassica napus(PGAZ) | CAC05658.1 | |||
甜瓜Cucumis melo(MPG2) | AAC26511.1 | |||
陆地棉Gossypium hirsutum(G9) | AAA82167.1 | |||
荔枝Litchi chinensisv(LcPG1) | AFW04075.1 | |||
番茄Solanum lycopersicum(TAPG1) | AAC28903.1 | |||
番茄Solanum lycopersicum(TAPG2) | AAC28904.1 | |||
烟草Nicotiana tabacum(PG1) | Q05967.1 | |||
桃Prunus persica(PRF5) | CAA54150.1 |
图2 BcPG17的信号肽(A)和跨膜结构(B)的预测 信号肽与成熟肽之间的分割位点可能位于第27和第28位氨基酸残基之间。
Fig. 2 Signal peptide(A)and transmembrane helices(B)prediction of BcPG17 The most likely cleavage site of signal peptide is between the position of 27 and 28.
图4 BcPG17及其在拟南芥中的同源基因At1g80170所在染色体区段的共线性分析
Fig. 4 Collinearity analysis of the chromosome segments where BcPG17 and its homologous gene At1g80170 in Arabidopsis thaliana are located
图5 BcPG17的表达特征和拟南芥数据库中At1g80170 ATH1基因芯片数据分析 Ⅰ~ Ⅴ分别代表花粉母细胞时期、四分体时期、单核小孢子时期、双核小孢子时期和三核成熟花粉期的花蕾。
Fig. 5 Analysis of the expression pattern of BcPG17 and the ATH1 GeneChip data of At1g80170 from Arabidopsis database Ⅰ ~ Ⅴ indicate the buds of pollen mother cell stage,tetrad stage,uninucleate microspore stage,binucleate microspore stage and mature pollen respectively.
图6 BcPG17在白菜开花期花茎横切片的原位杂交结果
Fig. 6 The in situ hybridization result of BcPG17 in cross-section inflorescence stem of Brassica campestris during flowering stage
顺式作用元件 cis-acting element | 序列 Sequence | 功能 Function | 数量 Number |
---|---|---|---|
CAAT-box | CAAAT | 启动子和增强子区域常见的顺式作用元件 Common cis-acting element in promoter and enhancer regions | 8 |
TATA-box | TATAAAT/TATA | 转录起始位点周围-30左右的核心启动子元件 Core promoter element around-30 of transcription start | 44 |
TCA-element | CCATCTTTTT | 水杨酸响应相关的顺式作用元件 cis-acting element involved in salicylic acid responsiveness | 2 |
ABRE | ACGTG | 脱落酸响应相关的顺式作用元件 cis-acting element involved in the abscisic acid responsiveness | 1 |
TGACG/CGTCA-motif | TGACG/CGTCA | 茉莉酸甲酯响应相关的顺式作用调节元件 cis-acting regulatory element involved in the MeJA-responsiveness | 4 |
GARE-motif | TCTGTTG | 赤霉素响应元件 Gibberellin-responsive element | 1 |
CAT-box | GCCACT | 与分生组织表达有关的顺式调控元件 cis-acting regulatory element related to meristem expression | 1 |
ARE | AAACCA | 厌氧诱导的关键顺式作用调节元件 cis-acting regulatory element essential for the anaerobic induction | 2 |
G-Box | CACGTT | 参与光响应的顺式作用调节元件 cis-acting regulatory element involved in light responsiveness | 1 |
Box 4 | ATTAAT | 光响应相关的保守DNA模块的一部分 Part of a conserved DNA module involved in light responsiveness | 4 |
Gap-box | CAAATGAA(A/G)A | 光响应元件的一部分 Part of a light responsive element | 1 |
TCT-motif | TCTTAC | 光响应元件的一部分 Part of a light responsive element | 1 |
AT1-motif | AATTATTTTTTATT | 光响应元件的一部分 Part of a light responsive element | 1 |
AE-box | AGAAACAA | 光响应元件的一部分 Part of a light responsive element | 1 |
GTGA-motif | GTGA | 花药特异基序 Anther specific motif | 4 |
E-box | CATTG | 绒毡层降解延迟蛋白(TDR)结合位点 Tapetum Degeneration Retardation(TDR)binding site | 2 |
表2 BcPG17启动子序列中的主要顺式作用元件
Table 2 The major cis-acting elements of BcPG17 promoter
顺式作用元件 cis-acting element | 序列 Sequence | 功能 Function | 数量 Number |
---|---|---|---|
CAAT-box | CAAAT | 启动子和增强子区域常见的顺式作用元件 Common cis-acting element in promoter and enhancer regions | 8 |
TATA-box | TATAAAT/TATA | 转录起始位点周围-30左右的核心启动子元件 Core promoter element around-30 of transcription start | 44 |
TCA-element | CCATCTTTTT | 水杨酸响应相关的顺式作用元件 cis-acting element involved in salicylic acid responsiveness | 2 |
ABRE | ACGTG | 脱落酸响应相关的顺式作用元件 cis-acting element involved in the abscisic acid responsiveness | 1 |
TGACG/CGTCA-motif | TGACG/CGTCA | 茉莉酸甲酯响应相关的顺式作用调节元件 cis-acting regulatory element involved in the MeJA-responsiveness | 4 |
GARE-motif | TCTGTTG | 赤霉素响应元件 Gibberellin-responsive element | 1 |
CAT-box | GCCACT | 与分生组织表达有关的顺式调控元件 cis-acting regulatory element related to meristem expression | 1 |
ARE | AAACCA | 厌氧诱导的关键顺式作用调节元件 cis-acting regulatory element essential for the anaerobic induction | 2 |
G-Box | CACGTT | 参与光响应的顺式作用调节元件 cis-acting regulatory element involved in light responsiveness | 1 |
Box 4 | ATTAAT | 光响应相关的保守DNA模块的一部分 Part of a conserved DNA module involved in light responsiveness | 4 |
Gap-box | CAAATGAA(A/G)A | 光响应元件的一部分 Part of a light responsive element | 1 |
TCT-motif | TCTTAC | 光响应元件的一部分 Part of a light responsive element | 1 |
AT1-motif | AATTATTTTTTATT | 光响应元件的一部分 Part of a light responsive element | 1 |
AE-box | AGAAACAA | 光响应元件的一部分 Part of a light responsive element | 1 |
GTGA-motif | GTGA | 花药特异基序 Anther specific motif | 4 |
E-box | CATTG | 绒毡层降解延迟蛋白(TDR)结合位点 Tapetum Degeneration Retardation(TDR)binding site | 2 |
图9 转化pBGWFS7.0-proBcPG17: GUS-GFP载体的T3代拟南芥阳性植株的GUS组织化学分析
Fig. 9 The histochemical GUS assays of T3 Arabidopsis thaliana plants transformed with pBGWFS7.0-proBcPG17:GUS-GFP vector
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