油茶自交、异交雌蕊脯氨酸含量及相关基因克隆表达分析

Proline contents and the related genes expression in the pistil of selfand cross-pollination in Camellia oleifera

为研究脯氨酸在油茶(Camellia oleifera)授粉受精过程中的生理作用及相关基因的表达状况,本研究以油茶‘华鑫’为材料,通过反转录PCR (RT-PCR)方法分别克隆出油茶脯氨酸合成途径相关的2个关键基因,即Δ1-吡咯啉-5-羧酸合成酶(P5CS)基因和Δ1-吡咯啉-5-羧酸还原酶(P5CR)基因,以及脯氨酸分解途径相关的2个关键基因,即脯氨酸脱氢酶(ProDH)基因和Δ1-吡咯啉-5-羧酸脱氢酶(P5CDH)基因。克隆的4个基因分别被命名为Co P5CS、Co P5CR、Co Pro DH和Co P5CDH,基因编码区长度分别为2 154、834、1 656和1 473 bp,分别编码538、277、511和490个氨基酸。进化树分析表明, 4个蛋白彼此间的同源性较低,而与茶树(C. sinensis)对应蛋白亲缘关系最近。实时荧光定量PCR结果表明:CoP5CS和CoP5CR在自异交2~96 h期间表达趋势基本一致,在授粉后36h达到最高值,自交授粉表达量明显高于异交授粉;CoP5CDH和CoProDH在授粉后0~36 h期间呈现明显下降的趋势,在36~60 h期间表达量基本没有变化,授粉后60 h有明显上升的趋势。自异交雌蕊游离脯氨酸的含量都在授粉后36 h达到最高值,自交雌蕊脯氨酸含量是异交的1.56倍,表明脯氨酸可能参与了油茶自交花粉管死亡的过程。该研究结果不仅为油茶自交不亲和机制提供新的参考材料,还拟为提高油茶坐果率提供理论支撑。

In order to investigate physiological role and the related genes expression of proline in pollination and fertilization in oiltea(Camellia oleifera), two biosynthesis genes(Δ1-pyrroline-5-carboxylate synthetase, P5 CS, and pyrroline-5-carboxylate reductase, P5 CR) and two decomposition genes(proline dehydrogenase, ProDH, and Δ1-pyrroline-5-carboxylate dehydrogenase, P5 CDH) were isolated from C. oleifera ‘Huaxin’ by the method of reverse transcription PCR. The four cloned genes were named as CoP5 CS, CoP5 CR, CoProDH and CoP5 CDH, respectively. The coding regions of 4 genes were 2 154, 834, 1 656 and 1 473 bp, encoding 538, 277, 511 and 490 amino acids, respectively. Phylogenetic tree analysis showed that the four proteins were less homologous to each other and closely related to the corresponding proteins of tea(C. sinensis). Real-time quantitative PCR showed that the expression of CoP5 CS is consistent with CoP5 CR in 2–96 h after self-and cross-pollination, and those of the two genes reached the highest value at 36 h, while the expression level in self-pollination was apparently higher than that in cross-pollination. CoP5 CDH and CoProDH showed an apparently downward trend during 0–36 h after pollination. There was almost no change in expression of CoP5 CDH and CoProDH during 36–60 h, but showing an apparently upward trend after 60 h. The content of free proline in the pistil reached the highest value at 36 h after both self-and cross-pollination, but the proline content after self-pollination pistil was 1.56 times than that after cross-pollination, which indicates proline may be involved in the death of pollen tube in C. oleifera. The results of this study would provide not only new reference materials for self-incompatibility mechanism, but also theoretical basis for improving the fruiting rate of C. oleifera.