摘要
核盘菌(Sclertinia sclerotiorum (Lib.)de Bary)引起的菌核病是油菜的一种最主要的病害,在中国和世界其他地方导致巨大的经济损失,至今未培育出高抗油菜品种,尚未找到有效的高抗材料。尽管化学农药的使用对菌核病具有一定的限制作用,但因农药残留以及缺乏及时准确的预测预报方法使得化学农药防效不高。目前,在重要的经济作物上已尝试采用基因工程来防治病害,然而,因为对菌核病的植物防卫分子机制尚不清楚,所以限制了利用基因工程方法来培育抗菌核病的油菜品种。
研究已表明,植物MAPKs (mitogen-activated protein kinases)在植物防卫中起作用,可能是病害防卫反应信号传导链中一种新的成分。本研究克隆鉴定了一个编码甘蓝型油菜(Brassica napus)MPK4(BnMPK4)的基因,并将该基因转入油菜,研究了转基因植株的抗病性及相关的分子机制。
首先克隆了BnMPK4的cDNA。DNAMAN软件分析发现这个cDNA的最终开放阅读编码框架编码373个氨基酸;BnMPK4与拟南芥、烟草、水稻和玉米的MPK4同源性分别为95%、85%、82%和81%,含有一个保守的氨基酸基序T201E202Y203,在其C-terminal extension含有一个共同的锚定区域CD domain。MAPK序列比对表明,BnMPK4属于TEY亚型B组。
用抗病信号分子甲基茉莉酸(MeJA)、水杨酸(SA)类似物苯丙噻重氮(BTH)或核盘菌致病因子草酸等化学物质分别处理油菜品种中双9号,采用荧光定量PCR方法测定BnMPK4在各种化学物质诱导后的表达量,结果表明,BTH和草酸快速诱导该基因上调表达,而MeJA抑制其表达。
对两个菌核病抗性不同的油菜品种接种S. sclerotiorum后,实时定量PCR实验结果显示,当核盘菌侵染6 h时,BnMPK4的转录水平在抗病品种中双9号中显著升高,在感病品种84039中下调表达;防卫素基因PDF1.2的表达也出现类似的现象,在抗病品种中上调,在感病品种中下调。
为了进一步研究BnMPK4的抗菌核病功能,构建了植物表达载体pG4A-BnMPK4,并且转化油菜品种84039。除草剂(Bar)筛选、PCR、Southern blotting和Northern blotting检测后,选择5棵过表达BnMPK4油菜转基因植株作进一步研究。离体接种和活体接种抗病鉴定实验表明,转基因植株明显增强菌核病抗性;病斑发展logistic分析显示,转基因植株对核盘菌抗性增强是由于侵染延迟和病斑扩展慢造成的;Trypan blue组织染色实验调查显示,BnMPK4的过表达抑制了核盘菌的生长。相对于非转基因植株,转基因植株也提高了对灰霉病菌Botrytis cinerea的抗性。
为了研究BnMPK4转基因植株的抗菌核病机理,采用定量RT-PCR调查转基因植株中植物防卫标记基因的表达变化。结果显示,当BnMPK4高表达时,PDF1.2(茉莉酸途径的标记基因)的表达水平显著提高,而PR-1(水杨酸途径的标记基因)下调表达。DAB(3,3-diaminobenzidine)组织染色实验表明,BnMPK4的过表达降低H2O2积累。外源H2O2处理转基因植株导致增强对菌核病和灰霉病的敏感性。这些结果表明MPK4正调控JA介导的防卫反应。本文提出BnMPK4和JA介导的防卫反应在油菜抗菌核病中具有重要的作用。
Sclertinia sclerotiorum (Lib.) de Bary causes a highly destructive disease in oilseed rape, resulting in a tremendous seed yield loss in China and other regions of the world. No immune or highly resistant cultivars of oilseed rape have been reported to date, and few genetic sources of resistance to the pathogen are available to breeders. Control of the disease depends heavily on application of fungicides to the crop, but this is expensive and can be ineffective due to the difficulties associated with applying sprays to thick canopies and a lack of suitable forecasting methods to enable timely application of fungicides. So far, attempts have been made to engineer disease resistance in economically important crop plants. However, the molecular basis of plant defense to this pathogen remains poorly understood, which restricts engineering resistance by transgenic approaches.
Plant mitogen-activated protein kinases (MAPKs) have been implicated in plant defense as a new component of the defense signaling pathways. In the study, we isolated a Brassica napus MPK4 (BnMPK4),and over-expressed the gene in oilseed rape to investigate its function in resistance to S. sclerotiorum and its resistance mechanism.
A cDNA clone encoding a mitogen-activated protein kinase Brassica napus MPK4 (BnMPK4) was isolated. DNAMAN program analysis show that the entire open reading frame of the cloned cDNA encodes a protein of 373 amino acid residues that exhibit 95%, 85%, 82% and 81% identity with Arabidopsis, tobacco, rice and maize MPK4. BnMPK4 contain conserved amino acid motif (T201E202Y203) and a CD domain in its C-terminal extension. The MAPK alignment using the ClustalX programs indicated that BnMPK4 belonged to B subgroup of The TEY subtype.
Using quantitative RT-PCR, we analyzed BnMPK4 expression changes in oilseed rape after treatment with the chemicals BTH, MeJA or oxalic acid. The results showed that BnMPK4 expression were promptly activated by BTH and suppressed by MeJA. The gene expression was promptly activated in OA treatment.
Using quantitative RT-PCR, we found that BnMPK4 along with PDF1.2 are inducible in a resistant line Zhangshuang 9, but consistently suppressed in a susceptible line 84039 after inoculation with S. sclerotiorum.
To further determine whether BnMPK4 functions in defense responses to the necrotrophic fungus S. sclerotiorum, we constructed a plant transformation vector pG4A-BnMPK4. The cv. 84039 was chosen as the recipient for transformation. Five independent transgenic oilseed rape lines over-expressing BnMPK4 were generated and confirmed by PCR, Southern blot and northern blot. These results showed that the gene BnMPK4 has been inserted into the genome of the recipient and its RNA level was much higher in all five transgenic lines than the untransformed.
Transgenic oilseed rape over-expressing BnMPK4 markedly enhanced resistance to S. sclerotiorum. Logistic analysis of lesion development after inoculation showed that enhanced resistance in the transgenic plants would be committed by both delay of lesion occurrence and slow lesion expansion rate. By using trypan blue staining, we found that over-expression of BnMPK4 inhibited the growth of S. sclerotiorum in oilseed rape. Inoculation experiment also indicated that transgenic plants gain enhanced resistance to Botrytis cinerea.
To further investigate mechanism of BnMPK4 in Resistance to Sclerotinia Sclerotiorum, analysis of expression changes of defense-marker genes were carried out by quantitative RT-PCR. Results showed high levels of BnMPK4 activity activate PDF1.2 but suppress PR-1 in the absence of induction by chemicals or pathogen treatment. By using 3,3-diaminobenzidine (DAB) staining, we found that transgenic plants constitutively decreased H2O2 production. H2O2 treatment to the transgenic plants resulted in enhanced susceptibility to both S. sclerotiorum and B. cinerea. These results supported the idea that MPK4 positively regulate jasmonic acid (JA)-mediated defense response, which plays an important role in resistance to S. sclerotiorum in oilseed rape.
引文
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