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作物学报 ›› 2020, Vol. 46 ›› Issue (8): 1146-1156.doi: 10.3724/SP.J.1006.2020.94198

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

BnaBZR1和BnaPIF4基因调控甘蓝型油菜弱光光效的机制

冯韬1,2,谭晖1,2,官梅2,官春云2,*()   

  1. 1遵义师范学院生物与农业科技学院, 贵州遵义 563000
    2湖南农业大学农学院/国家油料改良中心湖南分中心, 湖南长沙 410128
  • 收稿日期:2019-12-16 接受日期:2020-03-24 出版日期:2020-08-12 网络出版日期:2020-04-03
  • 通讯作者: 官春云
  • 作者简介:E-mail: 812298771@qq.com
  • 基金资助:
    国家重点基础研究发展计划(973计划)项目(2015CB150206)

Mechanism of BnaBZR1 and BnaPIF4 regulating photosynthetic efficiency in oilseed rape (Brassica napus L.) under poor light

FENG Tao1,2,TAN Hui1,2,GUAN Mei2,GUAN Chun-Yun2,*()   

  1. 1College of Biology and Agriculture, Zunyi Normal College, Zunyi 563000, Guizhou, China
    2College of Agronomy, Hunan Agricultural University/National Oilseed Crops Improvement Center in Hunan, Changsha 410128, Hunan, China
  • Received:2019-12-16 Accepted:2020-03-24 Published:2020-08-12 Published online:2020-04-03
  • Contact: Chun-Yun GUAN
  • Supported by:
    National Basic Research Program of China (973 Program)(2015CB150206)

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摘要：

甘蓝型油菜品系XY881和XY883是湘油15辐照诱变后连续自交筛选的2个种子含油量、光合效率和弱光敏感性等有明显差别的子代品系。分别从XY881和XY883中克隆了芸薹素唑抗性因子1 (brassinazole-resistant 1, BnaBZR1) 和光敏色素互作因子4 (phytochrome interacting factor 4, BnaPIF4)基因并进行了序列结构、表达和功能分析。结果表明, XY883的BnaBZR1和BnaPIF4基因存在结构变异, 引起表达和调控模式的差异。XY883中BnaBZR1的启动子具有124 bp的富含A/T的插入序列, 且XY883具有比XY881高的BnaBZR1表达, 并且在弱光和2,4-表油菜素内酯(2,4-BL)诱导下具有较少的表达变化。XY883中BnaPIF4的5'-UTR区域存在可变剪接, 形成长度分别为424 bp (U01)、239 bp (U02)和332 bp (U03)的3种5'-UTR, 在弱光和2,4-BL诱导下, XY883中3种可变剪接的BnaPIF4转录产物的变化不一致。将BnaPIF4的3个5'-UTR与CDS分别组合转化拟南芥后其表达在转录水平无明显差异, 但蛋白翻译存在明显差异, 表明BnaPIF4的5'-UTR变异影响其翻译过程。转BnaPIF4基因拟南芥出现株高增加、叶片狭长且光合作用下降的表型, 共转化BnaBZR1能减弱BnaPIF4造成的光合作用下降; 转BnaPIF4和BnaBZR1基因对油菜的影响与拟南芥相似, 但表型不如拟南芥明显, 表明BnaPIF4是油菜光合作用的负调控因子, 而BnaBZR1可对BnaPIF4的光合负调控产生拮抗; 这与XY881和XY883中两基因表达调控模式及其光合表型相吻合。

关键词: 甘蓝型油菜, BnaPIF4, BnaBZR1, 可变剪接, 插入突变

Abstract:

Double-low seed rape B. napus L. varieties XY881 and XY883 screened from the same parent Xiangyou 15 have significant differences in seed oil content, photosynthetic efficiency, and poor light sensitivity. Brassinazole-resistant 1 (BnaBZR1) and phytochrome interacting factor 4 (BnaPIF4) genes were cloned from XY881 and XY883, respectively, and their sequence structure, gene expression and gene function were analyzed. There were structural mutations in the BnaBZR1 and BnaPIF4 of XY883, causing differences in gene expression and regulation. The promoter of BnaBZR1 in XY883 had a 124 bp A/T-rich insertion mutation, and XY883 had a higher expression of BnaBZR1 than XY881, and fewer expression changes under low light and 2,4-epibrassinolide(2,4-BL) treatment. Splicing differences of BnaPIF4 in the 5'-UTR region were found in XY883, and the alternative splicing resulted in three 5'-UTRs of 424 bp (U01), 239 bp (U02), and 332 bp (U03). Under the induction of low light and 2,4-BL treatments the changed of three alternative spliced BnaPIF4 transcripts in XY883 had significant differences. The three 5'-UTRs combined with the CDS of BnaPIF4 were transformed into Arabidopsis thaliana. There was no significant difference in BnaPIF4 gene expression, but a significant difference in protein expression, indicating that the 5'-UTR mutation of BnaPIF4 affects protein synthesis. Transgenic BnaPIF4 Arabidopsis thaliana showed a phenotype with increased plant height, narrow leaves, and decreased photosynthesis. Co-transformation whit BnaBZR1 could attenuate the decrease in photosynthesis caused by BnaPIF4. The effects of BnaPIF4 and BnaBZR1 genes on Brassica napus L. were similar to those on Arabidopsis thaliana, while the phenotype was not as obvious as that of Arabidopsis thaliana, suggesting that BnaPIF4 is a negative regulator of photosynthesis in rapeseed, and BnaBZR1 can antagonize the negative regulation of photosynthesis by BnaPIF4, while is consistent with the regulation pattern and photosynthetic phenotype of the two genes in XY881 and XY883.

Key words: Brassica napus L., BnaPIF4, BnaBZR1, alternative splicing, insertion mutation

表1

基因克隆引物"

引物
Primer		 克隆产物
Product of cloning		 序列
Sequence (5'-3')
PIF4_mF mRNA of BnaPIF4 ATAGATCTCATCCCTAAAGA
PIF4_mR TATGTTCAAAAGATAGCCTTAG
BZR1_mF mRAN of BnaBZR1 GGAGAAGGAAAGAGAGATTC
BZR1_mR TTGAGAGAAACAAAATGGGC
PIF4_cF CDS of BnaPIF4 ATGGAACACCAAGGTTGGAG
PIF4_cR CTAACGGGGACCGTCGG
BZR1_cF CDS of BnaBZR1 ATGACGTCAGATGGAGCTACG
BZR1_cR TCAACCACGAGCTTTGCC
PIF4_pF Promoter of BnaPIF4 ATTGAAACCGATTGTAAGGA
PIF4_pR AGAAACAAAGGAGCATAAAG
BZR1_pF Promoter of BnaBZR1 GGTTATTTTCAAATAATGGATG
BZR1_pR CTTGAGCTCTTAGCCCTGTG
PIF4_uF 5'-UTR of BnaPIF4 ATAGATCTCATCCCTAAAGA
PIF4_uR GTCAGATCTCAGATTTGGAAAGC
PIF4_dF Full-length gene of BnaPIF4 ATAGATCTCATCCCTAAAGAAG
PIF4_dR TTTTGACAAACTAAACCAGG

表2

RT-qPCR引物"

引物
Primer		 基因
Gene		 序列
Sequence (5'-3')
ActF BnaActin GGTTGGGATGGACCAGAAGG
ActR TCAGGAGCAATACGGAGC
PIF4F BnaPIF4_cds CTGTGCTGTTGTGCTTAC
PIF4R AGTCTCTACATAAACCCATAGG
U1_F BnaPIF4_UTR_U01 CTGTGCTGTTGTGCTTAC
U1_R AGTCTCTACATAAACCCATAGG
U2_F BnaPIF4_UTR_U02 CTGTGCTGTTGTGCTTAC
U2_R TTCCTCTCACTTGCTCTC
U3_F BnaPIF4_UTR_U03 GAGAGCAAGTGAGAGGAA
U3_R CAGAAGCTGAAGTAGTAGAAG
BZR1F BnaBZR1 CTCTCATCTCCAACTTCCAA
BZR1R GACTCATCACACTCAGGTATA

图1

弱光和2,4-BL处理下XY881和XY883重要农艺性状 A: 种子含油量; B: 种子油酸含量; C: 单株总角果数; D: 每角果种子粒数; E: 成熟种子千粒重; F: 叶片RuBPCase含量。*P < 0.05, **P < 0.01。"

图2

XY881和XY883中BnaPIF4和BnaBZR1基因结构 A: BnaPIF4和BnaBZR1全长mRNA; B: BnaPIF4和BnaBZR1全长CDS; C: BnaPIF4和BnaBZR1启动子; D: BnaPIF4三种5'-UTR; E: BnaPIF4全长基因; F: XY881和XY883中BnaPIF4和BnaBZR1突变结构示意。1: XY881中BnaPIF4; 2: XY883中BnaPIF4; 3: XY881中BnaBZR1; 4: XY883中BnaBZR1; 01~03: XY883中BnaPIF4三种5'-UTRs; M1: 2K plus DNA marker; M2: 2K DNA marker。"

图3

BnaBZR1启动子插入突变、基因表达和BnaBZR1与BnaPIF4互作 A: BnaBZR1启动子插入突变序列分析; B: BnaBZR1和BnaPIF4酵母杂交分析; C: XY881和XY883中BnaBZR1时空表达; D: 弱光和2,4-BL处理下XY881和XY883中BnaBZR1表达。DAG表示种子萌发后天数。"

图4

BnaPIF4可变剪接对基因表达和翻译的影响 A: XY881和XY883中BnaPIF4时空表达; B: 弱光和2,4-BL处理下XY881和XY883中BnaPIF4表达; C: 含不同5'-UTRs重组pRI101-AN 载体结构; D: 转基因拟南芥中BnaPIF4 mRNA和蛋白表达; E: 转BnaPIF4拟南芥表型; F: 转BnaPIF4拟南芥RuBPCase含量。DAG表示种子萌发后天数。"

图5

转基因拟南芥和转基因油菜表型 A: 转基因拟南芥; B: 转基因拟南芥RuBPCase含量; C: 转基因拟南芥种子含油量; D: 转基因油菜; E: 转基因油菜中BnaPIF4和BnaBZR1表达; F: 转基因油菜RuBPCase含量。"

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