小麦条锈菌III型磷脂酰肌醇4-羟基激酶基因（PsPik1）的功能分析

doi:10.3864/j.issn.0578-1752.2015.16.006

摘要/Abstract

摘要： 【目的】克隆小麦条锈菌（Puccinia striiformis f. sp. tritici）III型磷脂酰肌醇4-羟基激酶基因（PsPik1），分析其在小麦条锈菌生长发育与致病过程中的作用。【方法】利用RT-PCR技术克隆PsPik1的cDNA序列，采用生物信息学技术分析该基因编码蛋白分子特征并构建进化树；运用实时荧光定量PCR技术，选用小麦条锈菌的延伸因子（elongation factor，EF）作为内参，明确该基因在小麦条锈菌夏孢子阶段和侵染小麦后6、12、18、24、36、48 h以及3、5、7、9、11 d共12个发育时期的表达特征；利用病毒介导的基因沉默技术（BSMV-host-induced gene silencing，BSMV-HIGS），分析沉默PsPik1后对小麦条锈菌生长发育及致病性的影响。通过传统酶切连接的方法构建重组载体BSMV:γ:PsPik1-as，利用体外转录技术获得BSMV α、β与γ3个组分，在小麦二叶一心期接种BSMV: γ:PsPik1-as、BSMV: γ:0-as与BSMV: γ:TaPDS-as。接种病毒后第10天，在小麦第4叶接种条锈菌CYR32，接菌后24、48、120 h分别取样，以检测PsPik1的沉默效率和进行组织学观察。样品经染色处理后，统计分析接菌后48 h沉默植株与对照植株中条锈菌菌丝分支数、吸器母细胞数与菌丝长度的差异，及120 h沉默植株与对照植株中条锈菌菌落面积的差异。在接菌后12 d观察条锈病发病情况并照相记录。【结果】PsPik1开放阅读框（open reading frame，ORF）全长4 485 bp，编码1 494个氨基酸，具有III型磷脂酰肌醇4-羟基激酶典型的脂激酶特有结构域（LKU）、钙神经传感蛋白（NCS1）结合位点、Hom2结构域和PI3K/PI4K超家族激酶结构域。聚类分析表明，PsPik1聚于PI4KIIIβ类群，PsPik1与柄锈菌属的Pik1亲缘关系较近，与子囊菌Pik1亲缘关系次之，而与动物及植物的PI4KIIIβ亲缘关系较远。实时荧光定量PCR结果表明，PsPik1在接种后6、12、18、24 h呈诱导上调表达趋势，分别为夏孢子时期表达量的2.66、7.04、3.42、2.96倍，其中在12 h其表达水平最高。接种后36 h至5 d，PsPik1的表达水平开始下降，一直低于夏孢子表达水平，分别为夏孢子表达量的60%、30%、16%、15%。接种后7—11 d，PsPik1的表达量开始升高，在接种后11 d恢复至与夏孢子相当的水平。PsPik1基因沉默后的组织学观察分析表明，与接种BSMV:γ:0-as（对照）相比，接种BSMV:γ:PsPik1-as植株中条锈菌组织分化与生长发育受到了不同程度的影响，表现在接菌后48 h菌丝长度和分支数目明显减小，接菌后120 h菌落面积明显减小。沉默效率检测结果显示，在接菌后24、48、120 h，接种BSMV:γ:PsPik1-as植株PsPik1的表达水平分别仅为对照植物的49%、49%、69%，表明PsPik1的表达明显地受到抑制。【结论】PsPik1可能参与了病菌侵染初期的侵染结构分化及寄生关系的建立，在条锈菌致病过程中发挥重要作用。

关键词: 小麦条锈菌')">

小麦条锈菌, III型磷脂酰肌醇4-羟基激酶, 实时荧光定量PCR, BSMV-HIGS

Abstract: 【Objective】The objective of this study is to clone a gene PsPik1 encoding type III PtdIns 4-kinase from Puccinia striiformis f. sp. tritici (Pst) and characterize its function during Pst growth and development. 【Method】The full-length cDNA of PsPik1 was obtained using RT-PCR. Bioinformatic methods were used to analyze molecular properties of PsPik1. Quantitative real-time PCR (qRT-PCR) was applied to examine the expression profiles of PsPik1 at twelve different development stages. The transcript level of PsPik1 was calculated by the 2^-^△△CT method with the EF1 gene of Pst as endogenous reference for normalization. BSMV-HIGS was employed to analyze the function of PsPik1 by knocking down its expression. Selected PsPik1 gene fragment was amplified by PCR from Pst cDNA using primers with restriction enzymes Not I and Pac I sites. Amplicon was ligated into the BSMV γ vector generating BSMV:γ:PsPik1-as. The native BSMV:γ:0-as and BSMV:γ:TaPDS-as were used as negative and positive control, respectively. After inoculation with BSMV at the two-leaf stage, wheat seedlings were maintained in a growth chamber and examined for symptoms at regular intervals. After 10-day post virus inoculation, the fourth leaves of inoculated wheat seedlings in the PsPik1 silencing group were inoculated with urediospores of Pst race CYR32. The fourth leaves were collected at 24, 48 and 120 hours post inoculation (hpi) for histological observation and detection of silencing efficiency. Stained leaf segments were examined with an Olympus BX-51 microscope for the numbers of hyphal branches and haustorial mother cells and lengths of infection hyphae at 48 hpi and for colony size at 120 hpi. The incidence of stripe rust was observed and recorded with camera at 12 days post inoculation (dpi).【Result】 Based on the bioinformatic analysis and RT-PCR, the full-length cDNA of PsPik1 was cloned. The open reading frame (ORF) of PsPik1 was 4 485 bp in length, encoding 1 494 amino acids containing lipid kinase unique domain (LKU), NCS1 binding site, Hom2 domain and PI3K/PI4K superfamily catalytic domain. Phylogenetic analysis indicated that PsPik1 was clustered into PI4KIIIβ group and more closely related to Pik1 from Puccinia group than to Pik1 from ascomyceteous fungi and PI4KIIIβ from animals and plants. qRT-PCR assays revealed that PsPik1 transcripts was up-regulated within 6-24 hpi and peaked at 12 hpi. PsPik1 expression at 6, 12, 18 and 24 hpi were 2.66-, 7.04-, 3.42- and 2.96-folds as high as that of resting urediospores. However, expression of PsPik1 was down-regulated between 36 hpi and 5 dpi with the lowest expression at 5 dpi when it was 6.67-fold lower than that of resting urediospores. From 7 to 11 dpi, PsPik1 expression was gradually increased and the amount of transcripts was as high as that of resting urediospores at 11 dpi. To determine cytological changes associated with fungal growth on plants, silenced for PsPik1 wheat leaves inoculated with race CYR32 were examined microscopically. The hypha length and the number of hypha branches of Pst at 48 hpi and the colony size per infection site at 120 hpi in BSMV:γ:PsPik1-as infected wheat leaves were much shorter than that in BSMV:γ:0-as infected plants (control). PsPik1 transcript level exhibited an average of 49%, 49% and 69% expression in BSMV:γ:PsPik1-as infected plants as high as that in control plants.【Conclusion】PsPik1 may play an important role in the regulation of plant penetration and infectious growth in Pst.

Key words: Puccinia striiformis f. sp. tritici, type III PtdIns 4-kinase, qRT-PCR, BSMV-HIGS

何付新，张阳，秦娟，马微，康振生，郭军. 小麦条锈菌III型磷脂酰肌醇4-羟基激酶基因（PsPik1）的功能分析[J]. 中国农业科学, 2015, 48(16): 3156-3165.

HE Fu-xin, ZHANG Yang, QIN Juan, MA Wei, KANG Zhen-sheng, GUO Jun. Functional Analysis of PsPik1 Encoding Type III Phosphatidylinositol 4-kinase in Puccinia striiformis f. sp. tritici[J]. Scientia Agricultura Sinica, 2015, 48(16): 3156-3165.

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