脯氨酸羟化酶GhP4H2在棉花纤维发育中的功能研究

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。

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

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.