DDRT-PCR技术分析籽粒苋镉胁迫下相关基因的差异表达

Analysis of Differentially Expressed Genes of Amaranthus hypochondriacus Under Cadmium Stress by DDRT-PCR

以镉污染修复植物籽粒苋(Amaranthus hypochondriacus)的根、叶、果为实验材料,利用DDRT-PCR技术分析籽粒苋Cd胁迫下基因表达水平的变化。结果表明:回收差异片段获得镉处理诱导的4个差异表达序列(EST-1、EST-2、EST-3和EST-4),经Blastn比对,EST-1与土人参26S核糖体RNA基因部分序列(GB|HQ843466.1)一致性为99%,EST-2与虎耳草26S核糖体RNA大亚基基因部分序列(GB|AF036498.1)一致性为93%,EST-3与大肠杆菌菌株TW14359 DNA聚合酶Ⅲα亚基基因(ECs0186)完整CDS(GB|EU906049.1)一致性为97%,EST-4与米曲霉RIB40β-葡萄糖苷酶基因(XM_001816779.2)一致性为99%;经Blastx比对,EST-1与假定蛋白MTR_5g051030[苜蓿](XP_003614386.1)氨基酸序列相似性为26%,EST-2与假定衰老相关蛋白[柏杉](GB|ACA30301.1)氨基酸序列一致性为98%,EST-3与DNA聚合酶Ⅲα亚基[大肠杆菌OP50](ZP_06935700.1)氨基酸序列一致性为100%,EST-4与β-葡萄糖苷酶[黄曲霉NRRL3357](XP_002383240.1)氨基酸序列一致性为99%;4个差异表达基因通过调控蛋白质代谢、DNA复制和糖代谢等途径来应答镉胁迫。

Amaranthus hypochondriacus has great potential for phytoremediation of cadmium（Cd）polluted soils. In this study, differential display reverse transcriptase polymerase chain reaction（DDRT- PCR） was used to screen differentially expressed genes in the roots,leaves and fruits of A. hypochondriacus under Cd stress. The novel bands were recovered and four differentially expressed c DNA fragments[EST（Expressed sequence tags）-1, EST-2, EST-3 and EST-4] were obtained. All ESTs had homology with known c DNA clones in the Genbank databases. The EST-1 was similar to partial sequences of Talinum paniculatum 26 S ribosomal RNA gene（GB|HQ843466.1）with an identity of 99%, while the EST-2 showed similarity to partial sequences of Saxifraga mertensiana large subunit 26 S ribosomal RNA gene（GB|AF036498.1）with an identity of 93%. The EST-3 was similar to complete cds of Escherichia coli strain TW14359 DNA polymerase Ⅲ alpha subunit（ECs0186） gene（GB |EU906049.1） and their identity was 97%. The EST-4 was similar to Aspergillus oryzae RIB40 beta-glucosidase A（XM001816779.2） and the identity was 99%. After Blast X comparison, EST-1 was similar to hypothetical protein MTR5g051030 [Medicago truncatula]（XP003614386.1）with an identity of 26%, EST-2 similar to putative senescence-associated protein[Cupressus sempervirens]（GB|ACA30301.1）with an identity of 98%, EST-3 similar to DNA polymerase Ⅲ subunit alpha[Escherichia coli OP50]（ZP06935700.1） with an identity of 100%, and EST-4 similar to beta-glucosidase[ Aspergillus flavus NRRL3357]（XP002383240.1）with an identity of 99%. These four differentially expressed genes were responsive to cadmium stress through regulating protein metabolism, DNA replication and glucose metabolism.