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作物学报 ›› 2021, Vol. 47 ›› Issue (7): 1239-1247.doi: 10.3724/SP.J.1006.2021.04217

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

脯氨酸羟化酶GhP4H2在棉花纤维发育中的功能研究

高璐1,2, 许文亮1,*()   

  1. 1华中师范大学生命科学学院 / 遗传调控与整合生物学湖北省重点实验室, 湖北武汉 430079
    2山西农业大学小麦研究所, 山西临汾 041000
  • 收稿日期:2020-09-22 接受日期:2020-12-01 出版日期:2021-07-12 网络出版日期:2020-12-29
  • 通讯作者: 许文亮
  • 作者简介:E-mail: 13613418576@163.com
  • 基金资助:
    本研究由国家自然科学基金项目资助(31970516);本研究由国家自然科学基金项目资助(31671735)

GhP4H2 encoding a prolyl-4-hydroxylase is involved in regulating cotton fiber development

GAO Lu1,2, XU Wen-Liang1,*()   

  1. 1Hubei Key Laboratory of Genetic Regulation and Integrative Biology / School of Life Sciences, Central China Normal University, Wuhan 430079, Hubei, China
    2Wheat Research Institute, Shanxi Agricultural University, Linfen 041000, Shanxi, China
  • Received:2020-09-22 Accepted:2020-12-01 Published:2021-07-12 Published online:2020-12-29
  • Contact: XU Wen-Liang
  • Supported by:
    This study was supported by the National Natural Science Foundation of China(31970516);This study was supported by the National Natural Science Foundation of China(31671735)

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摘要：

阿拉伯半乳聚糖蛋白(arabinogalactan proteins, AGP)在棉花纤维发育过程中发挥重要作用。AGP由富含羟脯氨酸的主链蛋白和大量II型阿拉伯半乳聚糖(arabinogalactan, AG)侧链组成, 其合成过程要经历2次翻译后修饰, 首先是氨基酸主链上的脯氨酸被脯氨酸羟化酶(prolyl-4-hydroxylases, P4H)羟基化, 随后在糖基转移酶(glycosyltransferases, GT)催化作用下将阿拉伯半乳聚糖或寡糖添加到羟脯氨酸残基上。我们前期利用P4Hs的抑制剂处理棉花离体胚发现, 棉纤维伸长受到抑制, 暗示P4H参与棉纤维生长发育过程。为深入研究P4H在棉纤维发育中的功能, 我们从棉花中分离鉴定了1个在棉纤维发育过程中高量表达的脯氨酸羟化酶基因GhP4H2。本研究分别构建了GhP4H2过表达和RNA interference (RNAi)载体, 通过农杆菌介导法转化棉花, 获得转基因植株, 发现过表达转基因棉花株系T1~T3代成熟棉纤维变短, AGP含量增加, AG多糖侧链也发生变化。对过表达转基因植株和野生型植株棉纤维的转录组分析表明, GhP4H2正调控包括AGP在内的细胞壁糖蛋白基因的表达。基于以上研究, 我们推测GhP4H2可能主要通过影响AGP糖链组分调控棉纤维生长发育。

关键词: 棉纤维, 阿拉伯半乳聚糖蛋白, 脯氨酸羟化酶

Abstract:

Arabinogalactan proteins (AGPs) perform crucial roles during cotton fiber development, and they are composed of a hydroxyproline (Hyp)-rich core protein and large type-II arabinogalactan (AG) moieties. AGPs are highly glycosylated proteins which involve two most important post-translational processes. First, the proline residues were hydroxylated by prolyl- 4-hydroxylases (P4Hs), then the Hyps were substituted with large arabinogalactan polysaccharides or small arabino- oligosaccharides by glycosyltransferases (GTs). Our previous work had shown that fiber elongation was repressed when in vitro-cultured cotton ovules were treated with inhibitors of P4Hs, suggesting that P4Hs were involved in cotton fiber development. In this study, GhP4H2 was detected to be relatively highly expressed at fiber elongation stage. Overexpression and RNAi-silencing vectors of GhP4H2 were constructed and transformed into cotton. Phenotypic analysis showed that GhP4H2 overexpressing fibers were significantly shorter compared with the wild type from T1 generation to T3 generation. In addition, AGP content had increased in GhP4H2 overexpression fibers. Immuno dot-blot analysis also showed that carbohydrate moieties of AGP had changed. Moreover, RNA-seq revealed that expression levels of genes encoding hydroxyproline (Hyp)-rich glycoproteins including AGPs were enhanced. Overall, these results indicated that GhP4H2 may regulate fiber development primarily through affecting AGPs composition.

Key words: cotton fiber, arabinogalactan proteins (AGPs), prolyl-4-hydroxylase (P4Hs)

表1

本研究所用引物"

引物
Primer		 正向序列
Forward sequence (5'-3')		 反向序列
Reverse sequence (5'-3')
Gh_D12G1409RT TAGAACCCAAGCTGGTCAGG TCGATTTAGACCCTGTCGATG
Gh_D01G1917RT AGATCTCATGCGCCGTCA AGGCAAAGAAAGAAACAAGTGTG
Gh_D07G2484RT TTTGCCTTTTTCTGTGAGCA GCTGGTTGGGATGAACACTT
Gh_D10G0598RT CTATGAGTGTGATTGGCGTACAGGTAA AATCCGTGGTCACGTTATATTTGG
Gh_D08G2475RT TAACCCACCAAGTGGGAAAC TGAGCTTAAGGGTGGTGCAT
Gh_A07G0433RT TGAAGAAGACGAAGAACCATCACCA AGAGCCAGAGCTTTCAACTGATGT
Gh_D05G0131RT TTAAGCCACCCATTGACCTC TCCTGAAAGAACCGGACAAC
Gh_D12G2178RT TCTTTTCCCTGGTGAGTGCT AACGGCAGCTTCAAGATCAG
GhGalT1RT TCCCTCCTCACCTTCGCCATTG CCTTGACGATCAGAAGGCATCCAC
GhGalT4RT GATGATACTTTGAAAATCGTTGCT ATTTTCTTCAAAGTTTCACCGCT
GhGalT6RT TCAGGAGTATGTACCCCAGCTTGC TGCAAATGAAGTCACATGTCGAC
GhGalT7RT CCTCACCAGATCAACAGCCCTCT CCGTTGAAGGCCCTGATGATC
GhP4H2RT ATCGGAATGTGCAAAGAAGG AGCCAGGAAGTTCTGCAGTT
GhUBI1RT CTGAATCTTCGCTTTCACGTTATC GGGATGCAAATCCTTCGTGAAAAC
GhP4H2 RNAiL1 CTTGGATCCGTGCAAAGAAGGGAATTG GGGTCTAGAAATAGCCAGGAAGTTCTG
GhP4H2RNAiL2 GGGGAGCTCGTGCAAAGAAGGGAATTG CTTGCGGCCGCAATAGCCAGGAAGTTCT
GhP4H2OE GGGTCTAGAATGGCTATTGAGAGGATTT CTTGTCGACTCAACATACTTTGCAGCTT

图1

GhP4H2的系统进化分析和GhP4H2基因表达谱 A: 棉花GhP4H2与拟南芥GhP4H蛋白系统进化关系; B: GhP4H2基因在棉花各组织的表达谱。1: 根; 2: 下胚轴; 3: 子叶; 4: 真叶; 5: 花瓣; 6: 花药; 7: 15 DPA胚珠; 8: 0 DPA纤维(含胚珠); 9: 3 DPA纤维(含胚珠); 10: 5 DPA纤维; 11: 9 DPA纤维; 12: 15 DPA纤维; 13: 20 DPA纤维。DPA: 开花后天数。误差线代表标准误差。"

图2

GhP4H2在野生型和转基因棉花株系中的表达分析 A: GhP4H2在不同株系5 DPA纤维中表达分析; B: GhP4H2在不同株系10 DPA纤维中表达分析。RiL16、RiL28表示2个独立的GhP4H2 RNAi株系; WT表示野生型; OEL10、OEL38表示2个独立的GhP4H2过表达株系。**表示在0.01水平差异显著。"

图3

GhP4H2转基因与野生型棉花成熟纤维表型分析 A: GhP4H2转基因棉花与WT成熟种子和纤维表型比较; B: GhP4H2转基因棉花与WT成熟纤维长度比较。**表示在0.01水平差异显著。株系名称缩写同图2。"

图4

GhP4H2转基因与野生型棉花AGP定量分析 A: 不同样品AGP特异反应色圈。B: 阿拉伯树胶标准曲线; 横坐标代表AGP浓度, 纵坐标代表对应的色圈面积。C: 不同样品AGP含量。0.1~0.6 (μg μL-1): 阿拉伯树胶浓度。**表示在0.01水平差异显著。株系名称缩写同图2。"

图5

AGP糖链抗原决定簇在GhP4H2转基因与野生型棉花纤维中的分布与丰度 -: PBS阴性对照。+: 1 mol L-1 arabic gum阳性对照。株系名称缩写同图2。"

图6

细胞壁相关差异表达基因分析 A: 细胞壁相关基因的主要类别; B: 糖蛋白相关基因的表达验证。PRP: 富含脯氨酸蛋白; AGP: 阿拉伯半乳聚糖蛋白; EXT: 伸展蛋白; PAL: 苯丙氨酸解氨酶。**表示在0.01水平差异显著。株系名称缩写同图2。"

图7

部分GhGalT基因表达分析 *、**分别表示在0.05和0.01水平差异显著。株系名称缩写同图2。"

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