青岛不同野生群体的单倍体和二倍体龙须菜的AFLP遗传多样性分析与比较

以青岛湛山湾的单倍体和二倍体龙须菜以及青岛台湾路的单倍体和二倍体龙须菜为实验材料,采用AFLP技术,分析了不同地理群体内单倍体和二倍体种群以及不同世代种群间的遗传变异参数。湛山湾单倍体种群的Na(1.169 9)、Ne(1.095 0)、H(0.058 2)、I(0.088 4)与二倍体种群的Na(1.333 3)、Ne(1.227 0)、H(0.129 7)、I(0.190 8),以及台湾路单倍体种群的Na(1.137 3)、Ne(1.089 9)、H(0.050 4)、I(0.074 6)与二倍体种群的Na(1.209 2)、Ne(1.138 6)、H(0.082 5)、I(0.121 9)相比较,可以看出不同世代倍性的龙须菜的遗传多样性是有差别的,同一地理群体的二倍体在遗传变异上高于单倍体;而且湛山湾-台湾路单倍体种群间的Ht(0.058 1)、Gst(0.066 9)、Nm(6.968 3)和二倍体种群间Ht(0.179 9)、Gst(0.327 1)、Nm(1.028 5)的数值也有很大差别,单倍体和二倍体是处于不同分化层次的2个种群,根据Wright对Nm的研究可知,单倍体种群是中度分化的种群,二倍体种群是高度分化的种群。通过分析可知,龙须菜的遗传多样性水平较低,这为育种策略的选择提供了依据。

链状亚历山大藻生长衰亡相关基因的筛选

采用抑制性消减杂交(Suppression subtractive hybridization,SSH)技术构建了衰亡期的链状亚历山大藻(Alexandriumcatenella)特异表达的cDNA文库。共获得800个克隆,利用巢式引物进行PCR筛选,最终确定阳性克隆556个。利用斑点杂交技术对这些阳性克隆进行差异筛选,获得差异表达克隆160个。测序后得到片段125个,经过归类,获得21种序列,其中6种在NCBI中经Blast,获得功能基因与其匹配,这些功能基因分别是CHK1类似检测点蛋白(checkpoint-like protein)、核酸外切酶复合体、谷氧还蛋白、Na+/K+ATPase、叶绿体中的一个开放阅读框和pG1蛋白,其他blast无同源序列,可能为新基因。推测链状亚历山大藻的衰亡过程可能涉及到DNA损伤、mRNA降解过程、氧化还原状态的变化、离子动态平衡的变化以及叶绿体一些生理状况的变化等过程。

龙须菜UDP-葡萄糖焦磷酸化酶基因的克隆及其表达与琼胶产量的关系

对红藻龙须菜(Gracilarialei maneiformis)UDP葡萄糖焦磷酸化酶(UGPase)基因的表达与不同藻株中藻体琼胶产量之间的相关性进行研究。通过PCR扩增的方法得到了龙须菜UGPase的基因序列。该基因cDNA序列的全长为2 200 bp,开放阅读框1 485 bp,编码494个氨基酸;龙须菜UGPase基因的氨基酸序列与其它物种的UGPase氨基酸序列相似性较高;该基因是单拷贝的,缺乏内含子;具有特殊的加尾信号。利用荧光实时定量PCR的方法检测UGPase基因相对表达量。结果表明,该基因的表达与琼胶含量存在显著相关性,即在高琼胶组藻株中的表达量显著高于低琼胶组藻株,对琼胶含量高低具有指示作用,显示了在产业化应用中的潜能。

Development of a SCAR marker for male gametophyte of Gracilariopsis lemaneiformis based on AFLP technique

The red alga Gracilariopsis lemaneiformis(Bory) is an economically valuable macroalgae. As a means to identify the sex of immature Gracilariopsis lemaneiformis, the amplifi ed fragment length polymorphism(AFLP) technique was used to search for possible sex- or phase-related markers in male gametophytes, female gametophytes, and tetrasporophytes, respectively. Seven AFLP selective amplifi cation primers were used in this study. The primer combination E-TG/M-CCA detected a specifi c band linked to male gametophytes. The DNA fragment was recovered and a 402-bp fragment was sequenced. However, no DNA sequence match was found in public databases. Sequence characterized amplifi ed region(SCAR) primers were designed from the sequence to test the repeatability of the relationship to the sex, using 69 male gametophytes, 139 female gametophytes, and 47 tetrasporophytes. The test results demonstrate a good linkage and repeatability of the SCAR marker to sex. The SCAR primers developed in this study could reduce the time required for sex identifi cation of Gracilariopsis lemaneiformis by four to six months. This can reduce both the time investment and number of specimens required in breeding experiments.