[關(guān)鍵詞]
[摘要]
目的 對鐵皮石斛Dendrobium officinale中糖基轉(zhuǎn)移酶基因DoUGT83A1進(jìn)行克隆,并對其進(jìn)行生物信息學(xué)及表達(dá)模式分析。方法 從鐵皮石斛“雁蕩山1號”葉組織中獲得DoUGT83A1基因,利用軟件進(jìn)行生物信息學(xué)分析,利用MEGA 11構(gòu)建系統(tǒng)發(fā)育樹,采用實時熒光定量PCR(qRT-PCR)檢測基因表達(dá)模式。結(jié)果 通過PCR擴(kuò)增得到1個DoUGT83A1基因(GI:1622023669),該基因的CDS序列為1 347 bp,編碼448個氨基酸。DoUGT83A1蛋白相對分子質(zhì)量為54 330,理論等電點(diǎn)為5.75,為親水性不穩(wěn)定蛋白,無信號肽和跨膜區(qū)域,含有41個磷酸化位點(diǎn),具有UDPGT和PSPG 2個保守結(jié)構(gòu)域。氨基酸序列分析表明DoUGT83A1蛋白與金釵石斛Dendrobium nobile的UGT83A1蛋白的同源性最高,相似度為96.36%。分子對接結(jié)果證明DoUGT83A1蛋白對山柰酚具有較好的催化活性。組織特異性表達(dá)分析結(jié)果顯示,DoUGT83A1在“雁蕩山1號”不同組織相對表達(dá)量為:葉>莖>花>根(白根、綠根尖)。順式作用元件分析結(jié)果表明,DoUGT83A1基因啟動子序列含有多個與非生物脅迫響應(yīng)相關(guān)的元件,且在低溫、缺磷和菌根共生誘導(dǎo)下顯著上調(diào)表達(dá),推測該基因可能參與調(diào)控鐵皮石斛菌根共生及非生物脅迫響應(yīng)過程。結(jié)論 成功克隆獲得鐵皮石斛DoUGT83A1基因,并進(jìn)行生物信息學(xué)分析和基因表達(dá)模式分析。為進(jìn)一步探究DoUGT83A1基因功能及其在菌根共生和非生物脅迫響應(yīng)中的作用機(jī)制提供理論依據(jù)。
[Key word]
[Abstract]
Objective To clone one glycosyl transferase gene named DoUGT83A1 from Dendrobium officinale and analyze its bioinformatics information and expression patterns. Methods The DoUGT83A1 gene was obtained from the leaf tissue of D. officinale ‘Yandangshan 1’. Online softwares were used for bioinformatics analysis. Phylogenetic tree was constructed using MEGA 11. Gene expression patterns were analyzed by qRT-PCR. ResultsThe DoUGT83A1 gene (GI: 1622023669) was cloned by PCR. The CDS sequence of the DoUGT83A1 gene was 1347 bp, encoding 448 amino acids. The DoUGT83A1 protein was a unstable hydrophilic protein, with no signal peptide or transmembrane region. The relative molecular weight of DoUGT83A1 was 54 330, and its theoretical isoelectric point (pI) was 5.75. DoUGT83A1 contained 41 phosphorylation sites and two conserved domains (UDPGT, PSPG). Amino acid sequence analysis showed that DoUGT83A1 had the highest homology with UGT83A1 protein of D. nobile, with a similarity of 96.36%. Molecular docking demonstrated the catalytic activity of DoUGT83A1 protein on kaempferol. Tissue-specific expression analysis showed that the relative expression levels of DoUGT83A1 varied across different tissues of ‘Yandangshan 1’ as follows: leaves > stems > flowers > roots (white roots, green root tips). The results of cis-acting element analysis showed that the promoter sequence of DoUGT83A1 contained multiple elements, which were related to abiotic stress response. And the expression was significantly up-regulated under the induction of low temperature, phosphorus deficiency and mycorrhizal symbiosis, which suggested that this gene may regulate the mycorrhizal symbiosis and abiotic stress response process of D. officinale. Conclusion The cloning, bioinformatics analysis and expression patterns analysis of DoUGT83A1 of D. officinale were completed, which provided a theoretical basis for further exploring the function of DoUGT83A1 and its mechanism in mycorrhizal symbiosis and abiotic stress response.
[中圖分類號]
R286.12
[基金項目]
國家自然科學(xué)基金項目(32102485);溫州市科技特派員專項項目(X2023018);樂清市農(nóng)業(yè)科技計劃項目(2023N002);浙江省農(nóng)業(yè)科學(xué)院樂清共同富裕產(chǎn)業(yè)研究院項目