The Dynamic Change of Gene Expression from Pinus massoniana in Response to Bursaphelenchus xylophilus Infestation
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摘要: 松材线虫病是一种严重危害马尾松生长的流行性病害，可导致马尾松枯萎死亡。为揭示该过程中基因的表达变化行为，本研究利用荧光定量PCR技术检测了松材线虫侵染不同天数下的马尾松较其对照样本中病原识别、抗逆调节、次生代谢、解毒作用及生长素响应等相关基因的表达变化。结果显示，除了与病原识别相关的CC-NBS-LRR抗性蛋白基因的表达随侵染天数的增加而增强之外，其他基因则在侵染2 d的马尾松样本中的表达水平最高，且明显高于未受侵染的对照马尾松样本，随后在侵染3天的马尾松中的表达又低于对照样本。此外，黄酮-3-羟化酶和细胞色素P450基因的表达随着侵染虫量的增加呈先上调后下调的变化方式。通过本研究初步揭示了马尾松响应松材线虫侵染的基因表达变化模式。Abstract: Pine wilt disease (PWD) is a devastating disease, which affects on the growth of masson pine (Pinus massoniana), often leading to withering and death. To reveal the change of gene expression pattern on this process, the gene dynamic expression from the P. massoniana infestation with nematode (Bursaphelenchus xylophilus) for 1, 2, and 3 days was studied. The genes, which were relevant to pathogen recognize, stress regulation, secondary metabolism, detoxification, and auxin-response were concerned. The results showed that most of the tested genes were presented highest expression level on the P. massoniana with infestation for two days, it was then down-regulated on these groups with 3-day infestation. Besides, the gene expression of CC-NBS-LRR resistance protein was continually increased in the treated P. massoniana compared with the corresponding control group. Additionally, genes expression of flavonoid 3-hydroxylase and cytochrome P450 were firstly up-regulated and then down-regulated on the P. massoniana respectively infected by graded increasing nematode qualities. The current findings preliminarily revealed the gene expression pattern on the P. massoniana in response to B. xylophilus infestation.
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Key words:
- Pinus massoniana /
- Bursaphelenchus xylophilus /
- gene expression /
- disease response
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图 1 松材线虫侵染不同天数下的马尾松较其对照样本的茎干总RNA
注：泳道1~3为受侵染马尾松茎干样本的总RNA；泳道4~6为对照马尾松茎干样本的总RNA。
Figure 1. Total RNA from the stems of P. massoniana after different days of B. xylophilus infestation and the corresponding control groups
图 2 松材线虫侵染不同天数下的马尾松较其对照样本中的病原识别和抗逆调节基因的表达差异
注：A为CC-NBS-LRR抗性蛋白基因；B为late embryogenesis基因；C为class Ⅶ chitinase基因；D为class Ⅵ chitinase基因。
Figure 2. The differentially expressed genes relative to pathogen recognition and resistance regulation from the stems of P. massoniana infected by B. xylophilus in comparison with the corresponding control groups
图 3 松材线虫侵染不同天数下的马尾松较其对照样本中的植保素合成相关基因的表达差异
注：A为flavonoid 3-hydroxylase基因；B为dihydroflavonol-4-reductase基因。
Figure 3. The differentially expressed genes involved phytoalexin synthesis from the stems of P. massoniana infected by B. xylophilus in comparison with the corresponding control groups
图 4 松材线虫侵染不同天数下的马尾松较其对照样本中的细胞色素P450基因的表达差异
注：A为cytochrome P450基因；B为cytochrome P450 mono-oxygenase superfamily基因；C为cytochrome P450 mono-oxygenase CYP7368基因。
Figure 4. The differentially expressed cytochrome P450 relevant genes from the stems of P. massoniana infected by B. xylophilus in comparison with the corresponding control groups
图 5 松材线虫侵染不同天数下的马尾松较其对照样本中的生长素响应基因的表达差异
注：A为auxin-responsive family protein基因；B为dormancy/auxin associated-like protein基因。
Figure 5. The differentially expressed genes involved in auxin-response from the stems of P. massoniana infected by B. xylophilus in comparison with the corresponding control groups
图 6 不同数量松材线虫侵染下的马尾松茎中的FH和CYP基因表达变化
注：A为FH基因；B为CYP基因。
Figure 6. Changes in the genes expression from the stems of P. massoniana after different quantities of B. xylophilus infestation
表 1 待测基因及其荧光定量PCR引物序列
Table 1. The candidate genes and their primers sequence using for qPCR
待测基因 上游引物序列 下游引物序列 dihydroflavonol-4-reductase 5′-GCGATGCCGCTATTGT-3′ 5′-TGAGGGCTTGCGAGAA-3′ CC-NBS-LRR resistance-like protein 5′-CCATAAGGATGTGAGAGCCA-3′ 5′-AAAGGTAGAAGTGCGAGAGG-3′ late embryogenesis abundant-like protein 5′-TGCCAAGTGGGACGATG-3′ 5′-TGGACGGTGACGCTGTTT-3′ class Ⅶ chitinase 5′-AGTTGAACCCTGCTCTGC-3′ 5′-GTGGAAACACCGACGAAG-3′ flavonoid 3-hydroxylase 5′-CAACGGCGTGAATGCTAAG-3′ 5′-AGAAGGAAGGCGTGGAGT-3′ class Ⅳ chitinase 5′-CGAGGGCAAGGGATTCTA-3′ 5′-ATTCCTGGCTGTTGATGGC-3′ auxin-responsive family protein 5′-ACGCTGCTGCTGTTCTCC-3′ 5′-CCAGGCTGTGGCATCTTC-3′ dormancy/auxin associated-like protein 5′-TTCAGGTCCCTTCTTTGC-3′ 5′-CTTTCGGTTTGTTTGCC-3′ cytochrome P450 mono-oxygenase superfamily 5′-AATCCGTCGTAGGCAACA-3′ 5′-GCCCGCCACATAGAAAT-3′ cytochrome P450 5′-GTCGGAAACCTCCACCAAC-3′ 5′-TAGGGACTGAGCCCAAGC-3′ cytochrome P450 monooxygenase CYP736B 5′-GTGGTCTTTGCTCCGTTAGGG-3′ 5′-AGCGTTGGCGTCATTCTGC-3′ actin 5′-CCTTGGCAATCCACATC-3′ 5′-TCACCACTACGGCAGAAC-3′ 
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