【摘要】：【目的】探明水楊酸(SA)對蘋果葉片基因轉(zhuǎn)錄調(diào)控的影響,鑒定SA信號途徑及其調(diào)控基因,為研究SA介導(dǎo)的抗病分子機制提供理論依據(jù)。【方法】生長30 d的‘嘎啦’組培苗葉片用2 mmol·L-1水楊酸(SA)處理12 h,以CTRL(0.2%乙醇)處理作為對照,利用Illumina Hi Seq TM 2000進行轉(zhuǎn)錄組測序,通過綜合的生物信息學(xué)分析(差異基因篩選、條件特異性分析、GO分類及KEGG富集分析等)篩選SA信號途徑的調(diào)控基因。克隆受SA特異性誘導(dǎo)表達基因的啟動子,利用蘋果細(xì)胞原生質(zhì)體轉(zhuǎn)化技術(shù),進行啟動子活性鑒定,確定對SA進行特異性響應(yīng)的核苷酸序列�！窘Y(jié)果】CTRL和SA處理分別獲得750 439 459 bp和751 596 153 bp的原始數(shù)據(jù),分別有44.77%和43.88%與‘金冠’蘋果基因組完全匹配。獲得3 329個顯著性差異基因,包括苯丙烷類、類黃酮等次生代謝物生物合成途徑的相關(guān)基因(如木質(zhì)素合成關(guān)鍵酶CAD、細(xì)胞色素P450、真菌抗性相關(guān)的β-1,3-葡聚糖酶等),調(diào)控植物病原菌互作途徑重要功能基因(鈣調(diào)蛋白Ca M、抗病蛋白RPM1、熱激蛋白HSP90、WRKY轉(zhuǎn)錄因子等)以及33個條件特異性誘導(dǎo)表達基因(NAC轉(zhuǎn)錄因子、NIMIN1、WRKY40、ERF轉(zhuǎn)錄因子等)。其中1 085個基因上調(diào),2 244個基因下調(diào)。差異基因主要涉及細(xì)胞過程、代謝過程和基因綁定、催化活性等;根據(jù)轉(zhuǎn)錄組學(xué)的結(jié)果,將SA響應(yīng)基因Md WRKY40的啟動子序列克隆到含有熒光素酶基因的表達載體中,置于熒光素酶基因的上游,轉(zhuǎn)化蘋果原生質(zhì)體細(xì)胞。SA處理的原生質(zhì)體細(xì)胞,熒光素酶的活性為未經(jīng)SA處理的20.6倍,而脫落酸(ABA)、茉莉酸(JA)、1-氨基環(huán)丙烷羧酸(ACC)對熒光素酶的活性沒有影響,說明該啟動子為蘋果中對SA進行特異性響應(yīng)的啟動子序列。不同區(qū)段的啟動子片段對SA響應(yīng)能力不同,從Md WRKY40翻譯起始位點ATG向上游500—1 000 bp只能響應(yīng)高濃度SA,而對低濃度SA不具有響應(yīng)能力,1 500 bp片段對高濃度SA響應(yīng)能力進一步顯著增強,對低濃度SA響應(yīng)也有微弱提高;而長度為2 000 bp的核苷酸片段無論對高濃度SA還是對低濃度SA都具有顯著響應(yīng)能力,且達到最強。與2 000 bp片段相比,2 500 bp的核苷酸片段沒有進一步增強啟動子片段對SA的響應(yīng)能力。超表達Md WRKY40蛋白對其自身的轉(zhuǎn)錄具有抑制作用�！窘Y(jié)論】2 mmol·L-1 SA處理所影響的基因主要參與了苯丙烷類、類黃酮的生物合成,植物病原菌互作及植物激素信號轉(zhuǎn)導(dǎo)途徑。位于Md WRKY40開放閱讀框上游的2 500 bp核苷酸序列,為對SA進行特異性響應(yīng)的核苷酸啟動子序列。在1 000—1 500 bp及1 500—2 000 bp具有顯著提高啟動子對SA敏感性的未知核苷酸序列,另外,Md WRKY40轉(zhuǎn)錄調(diào)控存在反饋抑制機制。
[Abstract]:[objective] to investigate the effect of salicylic acid (SA) on gene transcription in apple leaves, and to identify the SA signaling pathway and its regulatory gene. In order to provide theoretical basis for studying the molecular mechanism of disease resistance mediated by SA. [methods] the leaves of 'Gara' plantlets growing for 30 days were treated with 2 mmol L-1 salicylic acid (SA) for 12 h and CTRL (0.2% ethanol) as control. Illumina Hi Seq TM 2000 was used to sequence transcription sequence. The regulation genes of SA signaling pathway were screened by comprehensive bioinformatics analysis (differential gene screening, conditional specificity analysis, go classification and KEGG enrichment analysis). The promoter which was specifically induced by SA was cloned, and the promoter activity was identified by means of protoplast transformation of apple cells. [results] the original data of 750439 459 BP and 751,596,153 BP were obtained by CTRL and SA treatments, respectively, with 44.77% and 43.88% matching the genome of 'Golden Crown' apple, respectively. A total of 3,329 differentially expressed genes, including phenylpropane, were obtained. Genes related to the biosynthesis pathway of secondary metabolites such as flavonoids (such as CAD, Cad, P450, 尾 -1m3-glucanase associated with fungal resistance, etc.), and important functional genes (calmodulin) regulating the interaction of plant pathogens. Protein Ca M, resistance protein RPM1, heat shock protein HSP90 WRKY transcription factor, etc., and 33 conditional specific expression genes (NAC transcription factor NieMIN1, WRKY40, etc.). Among them, 1 085 genes were up-regulated and 2 244 genes were down-regulated. The differential genes are mainly involved in cellular processes, metabolic processes and gene binding, catalytic activity, etc. According to the results of transcriptome, the promoter sequence of SA responsive gene Md WRKY40 was cloned into the expression vector containing luciferase gene. When placed upstream of luciferase gene, the luciferase activity was 20.6 times higher than that of non-SA treated apple protoplast. The activity of luciferase was not affected by abscisic acid (ABA), jasmonic acid (JA) 1-aminocyclopropane carboxylic acid (ACC), which indicated that the promoter was the promoter of specific response to SA in apple. The response ability of different segments of promoter fragments to SA was different. From Md WRKY40 translation initiation site (ATG) to upstream, 500-1 000 BP could only respond to high concentration of SA, but not to low concentration of SA. The response of 1 500bp fragment to high concentration of SA was further enhanced, and the response to low concentration of SA was also slightly enhanced. The nucleotide fragment with a length of 2 000 BP was highly responsive to both high and low concentrations of SA. Compared with the 2 000 BP fragment, the 2 500 BP nucleotide fragment did not further enhance the response of the promoter fragment to SA. [conclusion] the genes affected by 2 mmol L-1 SA are involved in the biosynthesis of phenylpropanes, flavonoids, the interaction of plant pathogens and the signal transduction pathway of plant hormones. [conclusion] the overexpression of Md WRKY40 protein inhibits its own transcription. [conclusion] the genes affected by 2 mmol L-1 SA are mainly involved in the biosynthesis of phenylpropanes, flavonoids and plant pathogens. The 2 500bp nucleotide sequence located in the upper reaches of Md WRKY40 open reading box is a nucleotide promoter that responds specifically to SA. At 1 000-1 500 BP and 1 500-2 000 BP, the sequence of unknown nucleotides significantly increased the sensitivity of promoter to SA. In addition, there was a feedback inhibition mechanism in the transcriptional regulation of Md WRKY40.
【作者單位】： 山東農(nóng)業(yè)大學(xué)園藝科學(xué)與工程學(xué)院/作物生物學(xué)國家重點實驗室;
【基金】：國家自然科學(xué)基金(31272132) 山東省泰山學(xué)者工程啟動基金(tshw20120712)
【分類號】：Q943.2;S436.611

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