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作物学报 ›› 2021, Vol. 47 ›› Issue (3): 416-426.doi: 10.3724/SP.J.1006.2021.04108

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

甘蓝型油菜SnRK基因家族生物信息学分析及其与种子含油量的关系

唐婧泉(), 王南, 高界, 刘婷婷, 文静, 易斌, 涂金星, 傅廷栋, 沈金雄*   

  1. 华中农业大学作物遗传改良国家重点实验室 / 国家油菜工程技术研究中心, 湖北武汉 430070
  • 收稿日期:2020-05-15 接受日期:2020-08-19 出版日期:2021-03-12 网络出版日期:2020-09-08
  • 通讯作者: 沈金雄
  • 作者简介:E-mail: 519736700@qq.com
  • 基金资助:
    国家自然科学基金项目资助(31871654)

Bioinformatics analysis of SnRK gene family and its relation with seed oil content of Brassica napus L.

TANG Jing-Quan(), WANG Nan, GAO Jie, LIU Ting-Ting, WEN Jing, YI Bin, TU Jin-Xing, FU Ting-Dong, SHEN Jin-Xiong*   

  1. National Key Laboratory of Crop Genetic Improvement / National Engineering Research Center of Rapeseed, Huazhong Agricultural University, Wuhan 430070, Hubei, China
  • Received:2020-05-15 Accepted:2020-08-19 Published:2021-03-12 Published online:2020-09-08
  • Contact: SHEN Jin-Xiong
  • Supported by:
    This study was supported by the National Natural Science Foundation of China(31871654)

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摘要：

蔗糖非发酵相关蛋白激酶(sucrose non-fermenting-1-related protein kinase, SnRK)是植物中广泛存在的丝氨酸/苏氨酸蛋白激酶, 它们参与调控植物的信号传导、逆境响应和种子生长等生物学过程。为了解析甘蓝型油菜(Brassica napus L.)中SnRK基因家族的性质及其对种子含油量的影响, 本研究对BnSnRK基因家族的系统进化、基因结构、蛋白质理化性质、保守基序、蛋白质二级结构、顺式作用元件和亚细胞定位预测等进行分析, 并通过候选基因关联分析、单倍型分析和qRT-PCR筛选影响种子含油量的BnSnRK基因。结果显示, 鉴定得到92个BnSnRK成员, 分为3个亚族, 分布于甘蓝型油菜19条染色体上, 亚族之间蛋白质理化性质差异显著。多数基因拥有7~14个外显子; 同一亚族的motif分布情况更相似。BnSnRK家族主要在细胞质中表达, 蛋白质二级结构主要以α-螺旋和不规则卷曲为主。关联分析筛选出与油菜种子含油量相关的12个家族成员, 基因BnaC02g10730D可能负向调控甘蓝型油菜种子含油量, 基因BnaA07g12290D、BnaA10g22850D、BnaA08g18050D、BnaC04g44390D可能正向调控甘蓝型油菜种子含油量。不同环境之间种子含油量差异显著, 且与含油量相关的12个成员均含有MYB、MYC和ABA响应元件, 环境特异的含油量关联基因可能与植物非生物胁迫响应相关。本研究为BnSnRK基因功能验证及育种工作提供了理论参考。

关键词: 甘蓝型油菜, 种子含油量, 蔗糖非发酵相关蛋白激酶, 关联分析, 生物信息分析

Abstract:

Sucrose non-fermenting-1-related protein kinase (SnRK) is a widely existed serine/threonine protein kinase in plants, which is involved in the regulation of biological processes such as signal transduction, stress response and seed growth. In order to explore the mechamism of the SnRK gene families and its influence on the seed oil content of Brassica napus L., BnSnRK gene family system evolution, gene structure, physical and chemical properties of protein, conservative motif, protein secondary structure, cis-element and subcellular localization prediction were analyzed, and BnSnRK genes affecting oil content of seeds were screened by candidate genes association analysis, haplotype analysis and qRT-PCR. The results showed that 92 BnSnRK members were identified and divided into three subgroups, distributed on 19 chromosomes of Brassica napus L. The physical and chemical properties of proteins were significantly different among subgroups. Most genes had 7-14 exons; the motifs of the same subfamily were more similar in distribution. The BnSnRK family was mainly expressed in the cytoplasm. 12 family members related to the oil content were screened out by association analysis in rapeseed. Genes BnaC02g10730D might negatively regulate seed oil content, while genes BnaA07g12290D, BnaA10g22850D, BnaA08g18050D, and BnaC04g44390D might positively regulate seed oil content of Brassica napus L. There were significant differences in seed oil content under different environments, and 12 oil-related members all contained MYB, MYC and ABA response elements. Environment-specific oil-related genes might be related to plant abiotic stress response. This study provides a theoretical basis for BnSnRK gene functional verification and breeding.

Key words: Brassica napus, seed oil content, sucrose non-fermenting-1-related protein kinase, association analysis, bioinformation analysis

图1

基于BnSnRK的油菜种子含油量和脂肪酸组分的关联分析 图示为较宽松筛选条件下的关联分析结果汇总。点状标注共8层, 从内圈到外圈的表型为含油量、棕榈酸、硬脂酸、油酸、亚油酸、亚麻酸、芥酸、花生烯酸。正方形标注表示18碳类脂肪酸组分。"

附图2

italic>SnRK亚家族的理化性质比较"

附表1

BnSnRK蛋白二级结构分析"

结构类型
Structure type		 BnSnRK1 BnSnRK2 BnSnRK3
最小值
Minimum		 最大值
Maximum		 标准差
Standard deviation		 最小值
Minimum		 最大值
Maximum		 标准差
Standard deviation		 最小值
Minimum		 最大值
Maximum		 标准差
Standard deviation
α-螺旋
Alpha helix		 31.62 34.83 0.98 31.58 45.50 3.52 31.62 41.12 2.14
β-转角
Beta angle		 5.85 7.44 0.58 4.14 8.65 1.29 8.14 12.56 0.96
不规则卷曲
Irregular crimp		 40.70 44.92 1.30 34.05 43.09 2.50 29.95 39.92 2.50
延伸链
Extending chain		 15.82 18.96 0.91 13.01 20.35 1.83 15.73 21.10 1.22

附图3

BnSnRK基因家族成员富集分析"

附图1

SnRK基因家族亚细胞定位预测"

附图4

BnSnRK家族启动子顺式作用元件分析 MYB: MYB转录因子响应元件;MYC: MYC转录因子响应元件;MBS: MYB结合位点。"

图2

BnSnRK基因家族代表性成员的系统进化树、CDD保守结构和基因结构构成 BnSnRK基因家族分为3个亚族, 红色区域为SnRK1亚族, 蓝色区域为SnRK2亚族, 黄色区域为SnRK3亚族。"

附图5

BnSnRK部分成员的保守基序分析"

图10

附图6 E4环境含油量关联分析的曼哈顿图"

表1

重复检测到的油菜种子含油量关联位点"

基因名称
Gene name		 环境
Environment		 标记
Marker		 染色体
Chromosome		 LOD值
LOD value		 阈值
-log10 (P)		 表型贡献率
R2 (%)		 单倍型频率
Haplotype frequency
BnaA07g12290D E2 s5667 A7 3.79 4.53 7.79 0.42
BnaA10g22850D E2 s7970 A10 4.50 5.33 12.95 0.30
BnaA07g12290D E4 s5664 A7 3.42 4.14 1.99 0.48
BnaA07g12290D E4 s5675 A7 4.46 5.23 3.07 0.28
BnaA10g22850D E4 s8059 A10 5.44 6.26 6.70 0.11
BnaA10g22850D E4 s8018 A10 8.16 9.06 6.34 0.09
BnaA07g12290D E5 s5674 A7 5.07 5.87 4.16 0.28

附图7

单倍型分析箱线图 单倍型ACT和单倍型CTC分别为基因BnaA10g28850D含油量最低和最高的单倍型,在E2、E4、E5环境中用绿箱体表示;单倍型GGGT和TAAC分别为基因BnaA07g12290D的含油量最低和最高单倍型,在3个环境中用蓝色箱体表示;每个环境下的边框和线用不同颜色区分。"

附表2

单倍型分析的多重比较"

因变量: 含油量
Dependent variable: oil content		 (I) 单倍型
Haploid type		 (J) 单倍型
Haploid type		 平均差异
Average difference		 标准误
Standard error		 显著性
Significant
BnaA07g12290D GAGT GGAC -0.48587 0.59894 0.418
GGGT 0.31362 0.59408 0.598
TAAC -1.04697 0.56187 0.063
GGAC GAGT 0.48587 0.59894 0.418
GGGT 0.79949* 0.37903 0.035
TAAC -0.5611 0.32624 0.086
GGGT GAGT -0.31362 0.59408 0.598
GGAC -0.79949* 0.37903 0.035
TAAC -1.36059* 0.31722 0.000
TAAC GAGT 1.04697 0.56187 0.063
GGAC 0.5611 0.32624 0.086
GGGT 1.36059* 0.31722 0.000
BnaA10g22850D ACT CTC -2.57628* 0.44087 0.000
CTT -1.01843 0.85095 0.232
CTC ACT 2.57628* 0.44087 0.000
CTT 1.55785* 0.76008 0.041
CTT ACT 1.01843 0.85095 0.232
CTC -1.55785* 0.76008 0.041

图3

含油量相关候选基因在甘蓝型油菜种子中的表达分析 A: qRT-PCR分析BnSnRK基因家族中含油量相关候选基因在油菜幼嫩种子中的表达; B: 全部候选基因的相对表达。"

附图8

SnRK基因家族染色体位置分布图 图中BnSnRK1、BnSnRK2和BnSnRK3亚族的成员名称分别用绿、红、黑表示,没有成员分布的Random染色体未出现在图中。"

图4

甘蓝型油菜SnRK基因家族同源比对图(A)及甘蓝、甘蓝型油菜和白菜3个物种的共线联合比对关系(B) A图中浅蓝色背景线为甘蓝型油菜SnRK基因家族成员之间的共线性基因对, 彩色线条将可能与甘蓝型油菜种子含油量关联的基因成员高亮表示。B图中灰色背景线代表全基因组中的共线性区块, 高亮线条代表BnSnRK基因家族成员在3个物种间的共线性关系。"

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