薄壳山核桃CiSULTR2.1基因的克隆与表达分析

为探究薄壳山核桃CiSULTR2.1基因功能及表达特征,以一年生薄壳山核桃实生苗为材料,利用RT-PCR和PCR技术克隆CiSULTR2.1基因全长,利用RT-qPCR技术分析其在不同浓度硒酸盐处理下的表达情况。结果表明,克隆得到1 902 bp的序列,经分析此序列属硫转运蛋白基因CiSULTR2.1全长开放阅读框的cDNA,编码633个氨基酸。CiSULTR2.1蛋白分子质量为68 943.37 Da,为疏水性的非分泌蛋白,无信号肽,结构稳定,二级结构主要由α-螺旋(51.18%)、延伸链(12.64%)和无规则卷曲(31.28%)构成,包含硫转运蛋白典型的Sulfate-transp结构域(PF00916)和STAS结构域(PF01740)。RT-qPCR分析结果表明,CiSULTR2.1在薄壳山核桃幼苗根与叶中均有表达,40、80 μmol·L^-1硒酸盐处理下,12 h出现表达高峰,此后表达量明显下降;而0.5 μmol·L^-1硒酸盐处理下,CiSULTR2.1表达高峰出现时间延后至48 h。硒酸盐浓度过高时,CiSULTR2.1表达量显著下降且低于对照。CiSULTR2.1基因能够快速响应40、80 μmol·L^-1高浓度硒酸盐的诱导,而0.5 μmol·L^-1低浓度硒酸盐需要较长时间才能诱导其高表达。高浓度硒酸盐处理较长时间对植物产生毒性效应,植物对硒酸盐的吸收减少,硒含量降低。本研究结果为薄壳山核桃CiSULTR2.1基因功能及硒吸收机制的研究提供了理论依据。

Cloning and Expression Analysis of CiSULTR2.1 Gene in Pecan[Carya illinoinensis (Wangenh.) K. Koch]

In order to explore the function and expression characteristics of CiSULTR2.1 gene in the pecan, the full-length CiSULTR2.1 gene was cloned by RT-PCR and PCR and its expression under different concentrations of selenite were analyzed by using the annual pecan seedlings as materials. The ORF of the CiSULTR2.1 gene was 1 902 bp in length, encoding 633 amino acids. The molecular weight of the encoded protein was 68 943.37 Da, which was hydrophobic and structurally stable. It was a non-secretory protein with no signal peptide, whose secondary structure was consist mainly of α-helix (51.18%), extended chain (12.64%) and random coil (31.28%), including the typical domains of Sulfate-transp domain(PF00916) and STAS domain(PF01740) in Sulfur transporters. RT-qPCR analysis indicated that CiSULTR2.1 gene was expressed in roots and leaves of pecan seedlings. Under the treatment of 40 μmol·L^-1 and 80 μmol·L^-1 selenate, the expression reached peak at 12 h, then decreased significantly. Under the treatment of 0.5 μmol·L^-1 selenate, the peak of expression appeared at 48 h. When the concentration of selenate was too high, the expression of CiSULTR2.1 gene decreased significantly and was lower than the control. CiSULTR2.1 gene was able to respond rapidly to 40 and 80 μmol·L^-1 selenate, while it took a long time for low concentration of selenate at 0.5 μmol·L^-1 to induce high expression level of CiSULTR2.1. Treatment with high concentration of selenate for a long time had a toxic effect on plants, and the absorption of selenate by plants was reduced and the content of selenium was decreased. The study provides a theoretical basis for exploring the function of CiSULTR2.1 gene and selenium absorption mechanism in pecan.