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沙棘(Hippophae L.)为胡颓子科(Elaeagnaceae)沙棘属多年生灌木或小乔木,其果实油脂中高积累多种人体必需的脂肪酸(omega-3、omega-6和omega-7脂肪酸),在预防和治疗胃肠疾病、冠心病和癌症等方面具有显著疗效[1]。但较低的含油率(种子为7%~11%,鲜果肉为1%~5%)制约了沙棘油的高效开发利用[2-3],目前关于沙棘果肉油脂合成积累机制的研究较少,其中的关键基因及代谢途径尚未清晰[4]。
甘油三酯(Triacylglycerols,TAG)是沙棘油脂中的主要成分,其含量与果肉含油率密切相关[2, 5]。内质网上的3-磷酸甘油(glycerol-3-phosphate,G3P)连续组装来自于细胞质的acyl-CoA脂肪酸合成TAG[6],而G3P是由糖酵解的中间产物磷酸二羟丙酮(dihydroxyacetone phosphate,DHAP)被3-磷酸甘油脱氢酶还原而来[7],GPD1(glycerol-3-phosphate dehydrogenase)编码3-磷酸甘油脱氢酶,在碳水化合物和脂类代谢中起关键作用[8],是脂类合成的限速酶[9]。GPD1基因表达量增加2倍可使油菜(Brassica napus L)种子中G3P水平增加3~4倍,含油率提高40%[7, 10]。TAG的最后组装是以二酰甘油(diacylglycerol,DAG)为直接前体,经二酰甘油转移酶(diacylglycerol acyltransferases,DGAT)酰基化而成[11]。DGAT是影响脂类积累的限速酶[12-13],在许多植物体中编码两类非同源的DGAT基因(DGAT1和DGAT2)[14-15]。DGAT1基因突变体可限制野生型拟南芥(Arabidopsis thaliana (L.) Heynh.)的TAG合成,而过量表达DGAT1基因则可增加种子含油率11%~28%[13]。DGAT2基因在TAG合成过程中具有使DAG特异结合某些特殊脂肪酸的作用,如油桐(Vernicia fordii Airy-Shaw.)中的桐油酸(eleostearic acid)[16]。Chen等[17]将油桐的VfDGAT2基因导入红酵母(Rhodotorula glutinis)和拟南芥中,发现VfDGAT2基因表达量与总脂肪酸含量呈线性相关性。
阮成江等[18]研究表明TAG的生物合成与源基因“GPD1”和汇基因“DGAT”相关。GPD1基因是甘油酯合成通路中的关键限速酶基因,是油脂合成的“源基因”,具有调控TAG合成底物3-磷酸甘油含量的作用[9, 18];DGAT为甘油二酯酰基转移酶,是催化合成TAG的关键限速酶,也是植物油脂合成的“汇基因”[12, 18]。同时调控源汇基因的表达可提高植物种子含油率,解析二者的相互作用机制对提高油脂产量和品质具有重要作用。随后有学者将脂肪酸的生物合成形容为“推”,将脂肪酸组装合成TAG形容为“拉”[19],共同表达“推”和“拉”基因可明显提高油脂含量。因此,分析沙棘果肉油脂合成积累的源基因“GPD1”和汇基因“DGAT1和DGAT2”的表达与含油率的关系,对提高沙棘果肉油脂具有重要意义。
本研究以近缘高油品系‘TF2-36’和低油品系‘杂56’果肉为材料,分析不同时期的果肉含油率,利用qRT-PCR研究源基因“GPD1”和汇基因“DGAT1和DGAT2”的表达模式及其在高低油果肉中的表达差异,揭示高低油果肉的油脂合成积累与源汇基因表达的关系,为深入理解沙棘非种子组织(果肉)油脂合成提供科学依据。
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以‘TF2-36’(蒙古沙棘亚种)和‘杂56’(蒙古沙棘和中国沙棘杂交种)为试验材料,二者间基于ISSR标记分析的遗传相似系数为0.752[20],果实分别于2015年6月25日、7月6日、7月17日、7月28日、8月8日、8月19日、8月30日和9月10日采自黑龙江省农业科学院浆果研究所。各品系样品采自3株无性繁殖植株的多个部位,同植株果实混合,用锡纸包裹后置于液氮中速冻。样品运抵大连民族大学资源植物研究所,保存于-80℃冰箱备用。
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采用氯仿甲醇法[2, 21-22]测定不同时期沙棘果肉含油率:冷冻干燥的果肉粉末转移至玻璃试管中,加入甲醇和氯仿(均为色谱纯,Honeywell公司)漩涡混匀后超声30 min,上清液转移到新试管中,残渣用氯仿甲醇(体积浓度百分比2∶1)再次提取,合并的上清液加入其1/4体积的氯化钾溶液(质量浓度0.88%),收集下层液至玻璃样品瓶中,挥发至恒质量。含油率(%)=(m1-m2)/m×100;m1为油脂和玻璃样品瓶的质量/g;m2为玻璃样品瓶的质量/g;m为干燥样品粉末的质量/g,实验设3次生物学重复。
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参照柱式植物总RNA提取试剂盒(上海生物工程有限公司)方法提取沙棘果肉总RNA,根据PrimeScriptTM RT reagent Kit with gDNA Eraser试剂盒(大连宝生物公司)方法合成第一链cDNA[23]。本研究前期构建了沙棘种子、果肉、叶、茎和根转录组,获得了大量的功能基因注释以及差异表达基因信息。利用筛选获得的目的基因片段和PrimerQuest在线软件设计特异引物(表 1)。参照SYBR Premix Ex TaqTM Ⅱ(Tli RNaseH Plus)试剂盒(大连宝生物公司)方法和ABI7500 Real time PCR仪(美国Applied Biosystems公司)推荐程序进行qRT-PCR[11],以沙棘UBQ5为内参基因[4],采用2-ΔΔCt方法分析目的基因相对表达量[24]。实验设3次生物学重复。
表 1 基因名称及qRT-PCR引物
Table 1. Gene names and primers for qRT-PCR
基因名称
Gene name蛋白名称
Protein name引物序列primer sequences(5’→3’) 扩增长度The length of the amplification/bp 上游upstream 下游downstream UBQ5 泛素蛋白5 Ubiquitin 5 GGCGAGTTTGACCTTCTTCTT CCACCTTGTTCTTCGTCTCC 103 GPD1 甘油-3-磷酸酰脱氢酶1
Glycerol-3-phosphate dehydrogenaseAATCAACCGGACCAATGAAA CATGTTTGCATCCTTTGCTG 106 DGAT1 二酰甘油酰基转移酶1
Diacylglycerol acyltransferaseGCTGGTAGCATAATGTTGGTG AGGGAGATGTCCAACCCAAT 102 DGAT2 二酰甘油酰基转移酶2
Diacylglycerol acyltransferaseTCTTCTGGGGATTGTTTGGA CAAACTGACCATGTGCCTCA 132 -
利用SPSS 20.0软件进行单因素方差分析和LSD法进行差异性检验,采用EXCEL2010进行作图。
沙棘果肉发育期油脂合成积累的源汇基因协同表达
Coordinated Expression of Source and Sink Genes Involved in Lipid Biosynthesis and Accumulation During Seabuckthorn Pulp Development
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摘要:
目的 探讨沙棘果肉油脂合成积累与源汇基因表达的关系。 方法 以8个不同发育时期的近缘高油品系‘TF2-36’和低油品系‘杂56’果肉为材料,利用氯仿甲醇法测定含油率,采用qRT-PCR技术分析油脂合成源基因(GPD1)和汇基因(DGAT1和DGAT2)在近缘高低油果肉间的表达差异及其对油脂合成积累的影响。 结果 研究表明:(1)沙棘果肉含油率呈先上升后稳定趋势,‘TF2-36’的果肉含油率一直高于‘杂56’;(2)GPD1、DGAT1和DGAT2基因在‘TF2-36’果肉发育期间中均有明显高于‘杂56’的表达量峰值,但GPD1表达量峰值出现在油脂快速合成期,DGAT1和DGAT2表达量峰值出现在油脂稳定积累期。GPD1在发育前期高表达,促进合成更多的TAG前体G3P,而DGAT1和DGAT2在发育后期高表达,则促进了TAG的高积累。 结论 沙棘果肉高油脂积累源于源基因"GPD1"和汇基因"DGAT1和DGAT2"的协同高表达,研究结果为理解沙棘非种子组织(果肉)油脂合成机理提供了理论依据。 Abstract:Objective The objective of this study is to explore the relationship between lipid biosynthesis and source and sink genes' expression in seabuckthorn (Hippophae L.) pulp. Method Two close-related trains 'TF2-36'(with higher oil content) and 'Za 56' (with lower oil content) were selected as test samples. Their pulps were harvested in eight developmental stages. The oil content in pulp was tested by the method of chloroform methanol, and the differential expression of source gene 'GPD1' and sink genes 'DGAT1 and DGAT2' involved in lipid biosynthesis between high and low oil content lines were determined using qRT-PCR, and the effects of the three genes on lipid biosynthesis and accumulation were analyzed. Result (1) The oil contents in pulp of 'TF2-36' were higher than that of 'Za 56' at all stages, but it first increased, and then kept stable for two lines; (2) the peak values of GPD1, DGAT1 and DGAT2 expression in pulp of TF2-36 were significantly higher than that in 'Za 56' during pulp development. The peaks of GPD1 gene appeared in the period of rapid lipid biosynthesis, and the peaks of DGAT1 and DGAT2 genes appeared in the period of stable lipid accumulation. The high expression of source gene (GPD1) contributed to synthesis more G3P of TAG precursor in early stages of pulp development, but the high expression of sink genes (DGAT1 and DGAT2) accelerated high TAG accumulation in later stages of pulp development. Conclusion The high coordinated expression of source gene 'GPD1' and sink gene 'DGAT1 and DGAT2' resulted in the high lipid biosynthesis and accumulation in seabuckthorn pulp. These results provided basis for understanding lipid biosynthesis mechanism in seabuckthorn non-seed (pulp) tissue. -
Key words:
- Hippophae L.
- / lipid biosynthesis
- / source gene
- / sink gene
- / gene expression
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表 1 基因名称及qRT-PCR引物
Table 1. Gene names and primers for qRT-PCR
基因名称
Gene name蛋白名称
Protein name引物序列primer sequences(5’→3’) 扩增长度The length of the amplification/bp 上游upstream 下游downstream UBQ5 泛素蛋白5 Ubiquitin 5 GGCGAGTTTGACCTTCTTCTT CCACCTTGTTCTTCGTCTCC 103 GPD1 甘油-3-磷酸酰脱氢酶1
Glycerol-3-phosphate dehydrogenaseAATCAACCGGACCAATGAAA CATGTTTGCATCCTTTGCTG 106 DGAT1 二酰甘油酰基转移酶1
Diacylglycerol acyltransferaseGCTGGTAGCATAATGTTGGTG AGGGAGATGTCCAACCCAAT 102 DGAT2 二酰甘油酰基转移酶2
Diacylglycerol acyltransferaseTCTTCTGGGGATTGTTTGGA CAAACTGACCATGTGCCTCA 132 -
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