摘要
【目的】深入解析番茄mi R828的生物学功能,特别是参与番茄缺磷胁迫响应和花青素生物合成的调控作用。【方法】分别利用ps RNATarget和RLM-5′RACE预测和验证mi R828在番茄中的靶基因。用Meg Align和MEGA5对番茄Sl MYB7-like与部分R2R3 MYB转录因子氨基酸序列进行了多重比对和进化树分析。用q RT-PCR分析mi R828及其靶基因Sl Myb7-like在AC、Micro Tom和LA1996 3个不同番茄种质中的表达和mi R828在番茄(AC)中的组织特异性表达模式。以mi R828过表达转基因番茄和野生番茄为材料,设置正常供磷(+Pi,MS+KH2PO4 3.4 g·L-1)和缺磷(-Pi,MS+KCl 1.86 g·L-1)2个处理的培养试验。用q RT-PCR分析mi R828及其靶基因在缺磷胁迫下的表达模式。将野生型和mi R828过表达的转基因番茄植株缺磷培养15 d后,观察番茄植株地上部分和地下部分的表型差异,通过q RT-PCR分析mi R828、mi R828的靶基因Sl Myb7-like(SGN-U320618)和花青素合成相关酶基因(PAL、CHS、DFR、ANS、3GT和F3′H)的表达模式,应用分光光度计测量各样品的花青素含量。【结果】RLM-5′RACE剪切试验表明mi R828对靶基因Sl Myb7-like存在剪切调控作用,且剪切作用位点位于成熟mi R828的第10位和第11位核苷酸之间,从而验证了Sl Myb7-like是mi R828直接作用的靶基因。氨基酸序列多重比对及进化树分析显示番茄Sl MYB7-like与拟南芥At MYB7和金鱼草MYB330的序列相似性最高。Sl MYB7-like含有与花青素合成相关的保守氨基酸基序。mi R828在Micro Tom幼苗中的表达最高,在LA1996中的表达最低。相反,mi R828靶基因Sl Myb7-like在LA1996中的表达水平最高,在Micro Tom中的表达水平最低。q RT-PCR分析显示Sl Myb7-like的表达模式与mi R828相反,证明Sl Myb7-like被mi R828调控。正常供磷条件下,野生型番茄各组织中mi R828的表达量普遍较低,但在花芽、花和绿果中相对较高;mi R828过表达转基因番茄植株中各花青素合成相关基因�
【Objective】 In this study, the responses of mi R828 and its targets to phosphate deficiency and the role of mi R828 in anthocyanin biosynthesis under phosphate deficiency were investigated by using wild-type and mi R828 overexpressed transgenic tomatoes. 【Method】 The potential targets of mi R828 were predicted and validated by ps RNATarget and RLM-5′RACE, respectively. An alignment of deduced amino acid sequences of Sl MYB7-like with orthologs from Arabidopsis thaliana（At MYB7） and Antirrhinum majus（Am MYB330） was constructed using Meg Align of DNAStar. The phylogenetic tree of Sl MYB7-like and the selected R2R3 MYBs was constructed using neighbor-joining method using MEGA5. The expressions of mi R828 and Sl Myb7-like in AC, Micro Tom and LA1996 tomato seedlings were analyzed. The expression of mi R828 in different tissues/organs of tomato（AC） was analyzed by q RT-PCR. Wild-type and mi R828 overexpressed transgenic tomatoes were cultured under normal phosphate（KH2PO4 3.4 g·L-1） and phosphate deficiency（KCl 1.86 g·L-1） for 15 d. The phenotypic changes and the expression of mi R828, Sl Myb7-like（SGN-U320618）, several anthocyanin biosynthetic genes as well as the anthocyanin content were detected. 【Result】Sl Myb7-like was validated to be a direct target of mi R828. Protein sequence analysis showed that the Sl MYB7-like protein shares the highest homology with the Arabidopsis subgroup 4 MYB7（At MYB7）, and Snapdragon MYB330, having more than 80% sequence similarity. Phylogenetic analysis grouped Sl MYB7-like in a clade with At MYB7 and Am MYB330. Sl MYB7-like contains the conserved amino acid motif（[D/E]LX2[R/K]X3LX6L X3R/DLIVRLHSLLGNRWSLIAGR）, a signature feature common to subgroup 6 R2R3 MYBs（At MYB75/90/113） that are involved in anthocyanin biosynthesis. The highest abundance of mi R828 was detected in Micro Tom seedlings, where the transcript of its target gene（Sl Myb7-like） was accumulated the lowest. The expression of mi R828 in different tissues of tomato
出处
《中国农业科学》
CAS
CSCD
北大核心
2015年第15期2911-2924,共14页
Scientia Agricultura Sinica
基金
国家自然科学基金(31101555)
山西省青年基金(2011021032-2)
山西省留学基金(2011051)
山西省高等学校优秀青年学术带头人项目(2012)
山西人才引进与开发专项(614191)
山西省高等学校131人才项目
山西农业大学科技创新基金(2010021)
