干旱胁迫对东北红豆杉野生种与栽培种生理与次生代谢的影响

Effects of Physiology and Secondary Metabolism Between Wild and Cultivated Species of Taxus cuspidata under Environmental Stress

为探究干旱胁迫下东北红豆杉野生种与栽培种生理指标、次生代谢物含量以及相关基因表达量的差异,采用盆栽控水法模拟干旱胁迫环境,测定红豆杉叶片相对含水量、可溶性糖、可溶性蛋白、叶绿素、类胡萝卜素、类黄酮、丙二醛、游离脯氨酸含量及光合参数等指标;采用RT-PCR结合RACE技术克隆抗旱相关基因(TcDBTNBT、AP2、DREB、PsaK)。结果表明,干旱胁迫下,野生东北红豆杉光合参数,即净光合速率(P_(n))、蒸腾速率(T_(r))、气孔导度(G_(s))、叶绿素及相关基因(AP2、PsaK及TcDBTNBT)均呈下降趋势且始终高于栽培种,如干旱胁迫至第48天时,野生种P_(n)值约为栽培种的1.69倍,叶绿素含量约为栽培种的2.23倍;而野生种细胞间隙CO_(2)浓度(C_(i))、乳酸脱氢酶(LDH)、葡萄糖激酶(GCK)及细胞色素c(Cytc)则呈显著上升趋势且始终高于栽培种,C_(i)值在持续干旱胁迫至第48天时约为栽培种的1.32倍,LDH酶活性在第24天后急剧上升为栽培种的1.24倍,至第48天时约为栽培种的1.37;而东北红豆杉栽培种可溶性糖、可溶性蛋白、类胡萝卜素、类黄酮、脯氨酸、丙二醛、谷胱甘肽等次生代谢物含量以及DREB基因表达量均呈上升趋势且显著高于野生种,栽培种MDA含量干旱胁迫至第48天时,约为栽培种的1.36倍;栽培种谷胱甘肽(GSH)在第32天达到峰值,约为野生种的1.15倍,而后呈下降趋势,至第48天时,约为野生种的1.14倍,栽培种精氨酸和鸟氨酸转氨酶含量第16天时急剧上升,分别为野生种的1.05倍和1.19倍,而栽培种天冬氨酸含量在第24天达到峰值,约为野生种的1.07倍。综上所述,东北红豆杉通过糖类、氨基酸的显著增加来抵御干旱胁迫,磷酸戊糖途径是红豆杉体内重要的葡萄糖代谢途径之一,能量代谢方面野生东北红豆杉显著优于栽培种,从光合参数分析栽培种抵御干旱胁迫的能力不及野生东北红豆杉,而栽培种干旱胁迫应答的次级代谢产物如类黄酮等含量显著高于野生种。

In order to explore the differences of physiological indicators,secondary metabolite content and related gene expression between the wild and cultivars of Taxus cuspidata under drought stress,the pot water control method was used to simulate the drought stress environment,and the relative water content,soluble sugar,soluble protein,chlorophyll,carotenoids,flavonoids,malondialdehyde,free proline content and photosynthetic parameters were determined,the drought-related genes(TcDBTNBT,AP2,DREB and PsaK)were cloned by RT-PCR with RACE.The results showed that photosynthetic parameters,namely net photosynthetic rate(P_(n)),transpiration rate(T_(r)),stomatal conductivity(G_(s)),chlorophyll,and the AP2,PsaK and TcDBTNBT all decreased under drought stress,and the wild species was higher than the cultivated species consistently,for example,under drought stress for 48 d,the wild species Pn was about 1.69 times than that of cultivated species,and its chlorophyll content was about 2.23 times than that of cultivated species;the intercellular CO_(2) concentration(C_(i)),lactate dehydrogenase(LDH),glucose kinase(GCK)and cytochrome c(Cyt c)of the wild species increased significantly under drought stress,which were always higher than the cultivated species,Ci values were about 1.32 times than that of cultivated species at persistent drought stress for 48 d,and LDH enzyme activity increased sharply,which was about 1.24 times than that of cultivated species,1.37 times than that of cultivated species for about 48 d.However,the content of soluble sugar,soluble protein,carotenoids,flavonoids,proline,promaldialdehyde,glutathione and the expression of DREB gene of the cultivated species all showed an upward trend and was significantly higher than that of wild species,the MDA content of cultivated species was about 1.36 times than that of cultivated species under drought stress for 48 d,GSH content of cultivated species reached peak at 32 d and about 1.15 times than that of wild species,then it showed a downward trend,at 48 d,it was about 1.14 times than that of wild species,the arginine and nithine-δ-aminotransferase content increased sharply at 16 d,which was about 1.05 and 1.19 times than that of wild species separately,while the aspartic content reached peak at 24 d,which was about 1.07 times than that of wild species.In conclusion,wild species is significantly better than cultivated species in terms of energy metabolism,Taxus cuspidate resisted the drought stress by increasing their sugar and amino acid content,the pentose phosphate pathway is one of the important ways of glucose metabolism in it,the capability of the cultivated species to resist drought stress is less than wild species from photosynthetic parameters,while the secondary metabolites such as flavonoids from drought stress response in cultivated species are significantly higher than in wild species.