Identification of novel soybean oil content-related genes using QTL-based collinearity analysis from the collective soybean genome

Soybean is a global principal source of edible plant oil. As more soybean oil-related quantitative trait loci(QTLs) have been located in the collective genome, it is urgent to establish a classification system for these distributed QTLs. A collinear platform may be useful to characterize and identify relationships among QTLs as well as aid in novel gene discovery. In this study, the collinearity MCScan X algorithm and collective soybean genomic information were used to construct collinearity blocks, to which soybean oil-related QTLs were mapped. The results demonstrated that 666 collinearity blocks were detected in the soybean genome across 20 chromosomes, and 521 collinearity relationships existed in 231 of the 242 effective soybean oil-related QTLs. This included 214 inclusion relationships and 307 intersecting relationships. Among them, the collinearity among QTLs that are related to soybean oil content was shown on a maximum of seven chromosomes and minimum of one chromosome, with the majority of QTLs having collinearity on two chromosomes. Using overlapping hotspot regions in the soybean oil QTLs with collinearity, we mined for novel oil content-related genes. Overall, we identified 23 putatively functional genes associated with oil content in soybean and annotated them using a number of annotation databases. Our findings provide a valuable framework for elucidating evolutionary relationships between soybean oil-related QTLs and lay a foundation for functional marker-assisted breeding relating to soybean oil content.

An Integrated Quantitative Trait Locus Map of Oil Content in Soybean,Glycine max(L.) Merr.,Generated Using a Meta-Analysis Method for Mining Genes

Soybean is a major cash crop in the world, and its oil content was one of the very important traits. Therefore, the study of gene mapping for oil content in soybean is very important for breeding application. At present, at least 130 QTL loci for soybean oil content have been published; however, the mapping results of oil content were dispersed and a coalescent public map should be established to integrate the published QTLs, and to more efficiently mine genes based on the meta- analysis method of the bioinformatics tools. This study was to construct an integrated map of QTLs for soybean oil content and accelerate the application of bioinformation resource related to oil content improvement in the practice of soybean breeding. We collected information of 130 QTLs reported over the past 20 yr for soybean oil content and used the Software BioMercator 2.1 to project QTLs from their own maps onto a reference map, which was an early-integrated map constructed by Song （2004） for oil-content quantitative trait loci （QTLs） in soybean. Gene mining was performed based on the meta-analysis by running the local ver. GENSCAN and InterProScan. The confidence interval of QTLs was efficaciously narrowed using the meta-analysis method, and 25 consensus QTLs were mapped on the reference map. Using a local version of GENSCAN, 12 805 sequences in the consensus QTL intervals were predicted. With BLAST, these predicted sequences were aligned to gene sequences from the International Protein Index database using InterProScan locally. Thirteen predicted genes were in the class of the geme ontology （GO） accession （0006631）, which were involved in the fatty acid metabolic process. These genes were analyzed using BLAST at the NCBI website to examine whether they were related to oil content. Six genes were found in the oil-synthesis pathway. Twenty-five consensus QTLs and six genes were found in the oil-synthesis pathway. These results would lay the foundation for marker-assisted selection and mapping QTL precisely, and these genes will facilitate the researches on the gene mining of oil synthesis and molecular breeding in soybean.

Strategies to enhance cottonseed oil contents and reshape fatty acid profile employing different breeding and genetic engineering approaches

Cottonseed oil is the valuable byproduct extracted after seed cotton processing for lint. It confers a huge contribution to total vegetable oil production and ranked the 2nd to meet global edible oil requirements. Over centuries, breeders mainly focused to improve lint production and fiber quality. Now attention has been given to improve the cottonseed oil percentage, its functional and nutritional properties. However, these efforts are less than other major oilseed crops which left cottonseed oil market behind in term of consumer demand and kept cott on seed oil industry at vuln erable positi on. Con siderable progress has been made to alter the relative percentage of fatty acid composition still intensified efforts have been required to meet the global oilseed demand. The objective of this review is to explore the cotton germplasm variation for seed oil carrying potential, its utilization in suitable breeding programs, seed oil biosynthetic pathways, major genes, and QTLs linked to quantity and quality enhancement of oil and deployment of modern genomic tools, viz., gene silencing and transgenic development to ameliorate its nutritional properties.

Antimicrobial Effects of Plant Compounds against Virulent <i>Escherichia coli</i>O157:H7 Strains Containing Shiga Toxin Genes in Laboratory Media and on Romaine Lettuce and Spinach

Escherichia coli strains produce Shiga-toxins Stx-1 and Stx-2 that contribute to their virulence. The objective was to evaluate antimicrobial activities of plant essential oils (oregano, cinnamon, lemongrass), their active components (carvacrol, cinnamaldehyde, citral) and plant-extracts (green tea polyphenols, apple skin, black tea, decaffeinated black tea, grapeseed and pomace extracts) against E. coli O157:H7 strains containing Stx-1 and Stx-2 genes, as determined by Multiplex Polymerase Chain Reaction, in vitro and on leafy greens. Antimicrobials at various concentrations in sterile PBS were added to bacterial cultures (~3 - 4 logs CFU/ml), mixed thoroughly, and incubated at 37°C. Surviving bacteria were enumerated at 0, 1, 3, 5 and 24 h. The most effective essential oil (oregano oil;0.5%) and plant extract (green tea;3%) were evaluated against E. coli O157:H7 on romaine lettuce and spinach stored at 4°C for 7 days. Microbial survival was a function of the concentration of antimicrobials and incubation times. All antimicrobials reduced bacterial population to below detection levels in vitro;however, essential oils and active components exhibited greater activity than plant extracts. Oregano oil and green tea reduced E. coli O157:H7 on lettuce and spinach to below detection. Plant-based antimicrobials have the potential to protect foods against E. coli O157:

The molecular mechanism of WRINKLED1 transcription factor regulating oil accumulation in developing seeds of castor bean

The transcription factor WRINKLED1(WRI1),a member of AP2 gene family that contain typical AP2 domains,has been considered as a master regulator regulating oil biosynthesis in oilseeds.However,the regulatory mechanism of RcWRI1 in regulating oil accumulation during seed development has not been clearly addressed.Castor bean(Ricinus communis)is one of the most important non-edible oil crops and its seed oils are rich in hydroxy fatty acids,widely applied in industry.In this study,based on castor bean reference genome,three RcWRIs genes(RcWRI1,RcWRI2 and RcWRI3)were identified and the expressed association of RcWRI1 with oil accumulation were determined.Heterologous transformation of RcWRI1 significantly increased oil content in tobacco leaf,confirming that RcWRI1 activate lipid biosynthesis pathway.Using DNA Affinity Purification sequencing(DAP-seq)technology,we confirmed RcWRI1 binding with Transcription Start Site of genes and identified 7961 WRI1-binding candidate genes.Functionally,these identified genes were mainly involved in diverse metabolism pathways(including lipid biosynthesis).Three cis-elements AW-box([CnTnG](n)7[CG])and AW-boxes like([GnAnC](n)6[GC]/[GnAnC](n)7[G])bound with RcWRI1 were identified.Co-expression network analysis of RcWRI1 further found that RcWRI1 might be widely involved in biosynthesis of storage materials during seed development.In particular,yeast one hybrid experiments found that both AP2 domains within RcWRI1 were required in binding targeted genes.These results not only provide new evidence to understand the regulatory mechanism of RcWRI1 in regulation of oil accumulation during castor bean seed development,but also give candidate gene resource for subsequent genetic improvement toward increasing oil content in oilseed crops.

花生含油量的遗传基础与QTL定位研究进展

花生是我国重要的油料作物,含油量是花生重要的品质性状和育种目标。花生含油量每提高1个百分点,相当于增产2个百分点,油脂加工利润可提高7个百分点。培育高油高产花生品种是增加食用油供给和保障食用油供给安全的重要途径。本文概述了花生含油量表型鉴定的4种常用方法;阐述了花生含油量的遗传特性是多基因控制的数量性状,即受加性效应和显性效应的影响,也存在基因型和环境互作;总结了已报道的含油量QTL124个,表型变异解释率超过10%的主效位点有36个,分布在A03、A05和A08上的8个主效QTL可重复检测到;构建了一张花生含油量的一致性遗传图谱,A08染色体上33.59~50.24 Mb为热点区间;介绍了油脂合成及调控相关基因等方面的研究进展,以期为花生含油量遗传改良和高油品种培育提供理论指导。

棉籽蛋白与油分含量的遗传基础与QTL定位研究进展

棉花是重要的经济作物之一,在国民经济中占据重要地位。棉籽作为棉花生产中的重要产物,富含优质的蛋白质和油脂。充分挖掘利用棉籽中的蛋白和油分资源,能够在一定程度上缓解粮食安全问题。随着棉籽的综合利用价值不断被重视,有关棉籽营养品质遗传改良等的研究取得了长足的进步。本综述介绍了棉籽蛋白与油分含量鉴定的常用方法,概述了这些性状的遗传特性和影响因素;对棉籽蛋白、油分含量与纤维产量及品质性状之间的相关关系进行分析;收集整理已报道的335个油分含量数量性状位点(quantitative trait locus,QTL)和196个蛋白含量QTL构建了一致性物理图谱;对棉籽蛋白与油分相关合成途径和调控基因等方面的研究进展进行总结,并展望了棉籽营养品质生物育种未来的研究方向,旨在为棉籽营养品质的遗传改良提供参考。

采食高淀粉高油日粮奶牛的泌乳性能、乳成分变化及乳腺差异表达基因筛选

【目的】研究高淀粉高油日粮对奶牛泌乳性能和乳腺组织转录组的影响,探索乳脂下降综合征(Milk fat depression,MFD)的分子机制。【方法】选用8头荷斯坦泌乳奶牛,随机分为对照组和处理组(4头/组),采用2×2反转试验设计,分为2期,每期23 d,期间2组对调。每期的前16 d,对照组奶牛饲喂低淀粉低油的基础饲粮,对照组日粮的泌乳净能为6.78 MJ/kg;处理组奶牛在基础饲粮基础上添加266 g/kg(DM基础)细粉碎玉米和46 g/kg(DM基础)大豆油;后7 d 2组奶牛均喂基础饲粮,处理组日粮的泌乳净能为7.66 MJ/kg。分别于每期的第1、4、7、10、13、16、19和22天测定产奶量和乳成分,第16天采集乳腺组织用于检测转录本的变化。【结果】与对照组相比,处理组第13、16天奶牛的干物质采食量和产奶量显著降低(P<0.05);饲喂高淀粉高油饲粮7 d后乳脂率开始降低,其中,第10、13、16和19天的乳脂率和乳脂产量显著降低(P<0.05);第13、16和19天的乳蛋白、乳糖和非脂固形物含量显著提高(P<0.05)。在奶牛乳腺对照组和处理组中共检测到235个显著差异表达的基因,其中,64个上调、171个下调。GO分析表明,差异表达基因主要与炎症反应、α−氨基酸代谢、有机氮化合物代谢、细胞发育、脂质生物合成和脂肪细胞分化等相关;KEGG分析表明,差异表达基因主要与轴突导向、NF-κB信号通路、钙信号通路等相关。结合差异表达基因与泌乳性能的相关性分析,本研究筛选出了部分与脂质调控相关的基因作为研究奶牛MFD状态下脂质代谢的候选基因,包括在乳腺中下调表达的FGFR4、VDR、HTR2B、CCL21和TYRP1。【结论】高淀粉高油饲粮饲喂奶牛降低了干物质采食量、产奶量和乳脂率,提高了乳蛋白、乳糖和非脂固形物含量,下调了奶牛乳腺中脂肪酸合成相关基因的表达。本研究为科学配制日粮和研究MFD的分子机制研究提供了数据参考。

石油污染土壤微生物修复技术及机理研究进展

综述了环境微生物技术在石油污染土壤修复中的应用方法,介绍了人为干预提高石油降解微生物治理油污土壤的原位和异位修复技术和微生物去除石油组分的作用机理。针对微生物修复的研究热点多为石油降解微生物的选取及多种微生物的复合菌对石油组分的去除效果,但对微生物技术的实际应用和机理研究相对较少。提出了未来微生物技术修复石油污染土壤的研究和发展方向,包括特定材料对微生物的固定化、利用宏组学与示踪技术进一步明确微生物的降解机理和质粒拼接手段构建高效基因工程菌。

Suppressive action of garlic oil on growth and diferentiation of human gas-tric cancer cell line BGC-823

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Dynamic analysis of gene expression and determination of chemicals in agarwood in Aquilaria sinensis

Agarwood is the resinous heartwood of Aquilaria species.However,low yields and high costs of existing stimulation methods have led to the need for new techniques to produce agarwood rapidly and effectively.We developed a biological agarwood-inducing technique(Agar-Bit)that produces high yields and quality within 6 months.To better understand agarwood formation by Agar-Bit,dynamic gene expressions of key synthetases pathways of sesquiterpenes and chalcone-related enzymes at different times were determined after both Agar-Bit and the traditional burning chisel drilling(BCD)stimulation on Aquilaria sinensis trees.The qRT-PCR results show that some characteristic synthase genes were expressed at greatly different levels and times compared with the controls.For the Agar-Bit technology,main changes were after the 3rd or 5th month,while BCD expression clearly changed at the 5th month.Essential oils and total chromone contents were simultaneously determined.In the Agar-Bit group,both were higher and similar to natural levels.The Agar-Bit methodology is a new option for producing agarwood as demonstrated by genetic and chemical aspects.The differences in gene expression within 6 months for both groups indicates that the mechanisms of the two methods are different.These findings provide information on genetic variation during the process of agarwood formation.

Genome-wide analysis of the B3 transcription factors reveals that RcABI3/VP1 subfamily plays important roles in seed development and oil storage in castor bean(Ricinus communis)

The B3 transcription factors(TFs)in plants play vital roles in numerous biological processes.Although B3 genes have been broadly identified in many plants,little is known about their potential functions in mediating seed development and material accumulation.Castor bean(Ricinus communis)is a non-edible oilseed crop considered an ideal model system for seed biology research.Here,we identified a total of 61 B3 genes in the castor bean genome,which can be classified into five subfamilies,including ABI3/VP1,HSI,ARF,RAV and REM.The expression profiles revealed that RcABI3/VP1 subfamily genes are significantly up-regulated in the middle and later stages of seed development,indicating that these genes may be associated with the accumulation of storage oils.Furthermore,through yeast one-hybrid and tobacco transient expression assays,we detected that ABI3/VP1 subfamily member RcLEC2 directly regulates the transcription of RcOleosin2,which encodes an oil-body structural protein.This finding suggests that RcLEC2,as a seed-specific TF,may be involved in the regulation of storage materials accumulation.This study provides novel insights into the potential roles and molecular basis of B3 family proteins in seed development and material accumulation.

Identification and Expression Analysis of the Phosphoenolpyruvate Carboxylase Gene Family in Peanut (Arachis hypogaea L.)

Phosphoenolpyruvate carboxylase （PEPC） is widely distributed in plants and bacteria, and catalyzes the carboxylation of phosphoenolpyruvate to form oxaloacetate and inorganic phosphate. To investigate the molecular mechanisms of the regulation and control of peanut oil, with the degenerated primers and RACE-PCR approach, five PEPC genes were cloned from peanut, and designated as AhPEPC1, AhPEPC2, AhPEPC3, AhPEPC4, and AhPEPC5, respectively. The structure and phylogenetic analysis of PEPC protein indicated that AhPEPC1-4 genes encoded a typical plant-type PEPC-enzyme, and AhPEPC5 a bacterial-type. By real-time quantitative RT-PCR approach the expression pattern of each gene was detected in various tissues of normal and high oil-content peanut varieties. It was found that there was a lower expression level of AhPEPCs genes except for the AhPEPC2 in high-oil peanut than normal-oil peanut line. The results provide some fundamental information for the further investigation of plant PEPC proteins and their role in regulation of oil-content in peanut seeds.

Evaluation on Genetic Quality and Traits of Rice Landraces in the Taihu Lake Area

In this paper,with 511 rice landraces in the Taihu Lake area as test materials,we select 19 starch synthesis-related intragenic molecular markers to detect the genetic quality of starch,and compare them with 86 bred varieties. The results show that the average polymorphic information content( PIC) of japonica landraces is 0. 1726,slightly higher than the average PIC( 0. 1101) of the bred japonica rice varieties.Based on Nei's genetic distance between materials,UPGMA method is used for clustering,and all study materials are divided into 6 groups.Group I mainly includes indica rice,the bred japonica rice varieties are mainly concentrated in the first half of Group II and Group III,and the japonica landraces are mainly concentrated in the second half of Group III,and Group Ⅳ,Ⅴ,Ⅵ. Both of them are in different regions,and there has been genetic differentiation. According to the national standard of high quality rice,it is found that many rice landraces in the Taihu Lake area have good quality and traits,and these varieties can be used for future high quality breeding.

RNA/DNA Ratio and LPL and MyoD mRNA Expressions in Muscle of Oreochromis niloticus Fed with Elevated Levels of Palm Oil

Palm oil is of great potential as one of the sustainable alternatives to fish oil(FO) in aquafeeds.In this present study,five isonitrogenous diets(32% crude protein) with elevated palm oil levels of 0%,2%,4%,6% and 8% were used during an 8-week feeding trial to evaluate its effects on RNA/DNA ratio and lipoprotein lipase(LPL) and Myo D m RNA expressions in muscle of Oreochromis niloticus.The results showed that RNA,DNA content as well as ratio of RNA to DNA were significantly affected(P < 0.05),in each case the highest was recorded in fish group subjected to 6% palm oil level.There was a strong positive correlation between nucleic acid concentration(RNA concentration and RNA:DNA ratio) and specific growth rate(SGR),protein efficiency ratio(PER),while a negative correlation existed between nucleic acid concentration(RNA concentration and RNA:DNA ratio) and feed conversion ratio(FCR).The m RNA expressions of LPL and Myo D in muscle were not significantly affected by the different palm oil levels,although the highest expression was observed in fish fed with 6% palm oil level.There also existed a strong positive correlation between the m RNA expression of LPL,Myo D and SGR,PER,while their correlation with FCR was negative.In conclusion,elevated palm oil affected the RNA,DNA concentration as well as RNA/DNA ratio significantly,although the m RNA expression of LPL and Myo D were not affected significantly by elevated palm oil levels.

不同含油量花生品种中二酰甘油酰基转移酶基因的表达分析

二酰甘油酰基转移酶(DGAT)在植物油脂合成过程中发挥重要作用。本研究利用实时荧光定量PCR检测方法,以低油低油酸品种花育17号、高油高油酸品种花育910和高油低油酸品种花育918为试材,分析了不同含油量花生品种不同发育时期种子中AhDGAT1-1、AhDGAT1-2和AhDGAT3-3基因的表达情况。结果表明:AhDGAT1-1基因表达峰值出现在低油低油酸品种和高油高油酸品种的荚果发育初期(下针后30天),而在高油低油酸品种中则出现在荚果发育中期(下针后40天);AhDGAT1-2基因的表达峰值出现在低油低油酸品种的荚果发育初期、高油高油酸品种和高油低油酸品种的籽仁油脂积累快速期;AhDGAT3-3基因在3个花生品种中的表达峰值均出现在荚果发育的中后期。推测AhDGAT1-1、AhDGAT1-2和AhDGAT3-3基因表达量的增加有利于花生油脂的合成与积累,但其起作用的时期存在种质差异。本研究结果可为花生高油、高油酸品种的选育提供理论依据。

奇异南星挥发油通过miR-762/NF2轴对甲状腺癌细胞恶性生物学行为的影响

目的探讨奇异南星挥发油(ADVO)对甲状腺癌细胞恶性生物学行为的影响及可能作用机制。方法用含不同浓度ADVO培养液培养甲状腺癌TPC-1细胞48 h,CCK-8法检测细胞存活率,筛选最适作用浓度;TPC-1细胞分为空白组(常规培养)、ADVO组(60μg/mL ADVO)、miR-762 mimics组(转染miR-762 mimics)、miR-762 mimics NC组(转染miR-762 mimics NC)、ADVO+miR-762 mimics组(60μg/mL ADVO+转染miR-762 mimics)、ADVO+miR-762 mimics NC组(60μg/mL ADVO+转染miR-762 mimics NC)并培养,行CCK-8法、流式细胞术、Transwell小室实验;双荧光素酶报告基因实验验证miR-762与2型神经纤维瘤(Neurofibromatosis type 2,NF2)基因的靶向关系;RT-qPCR法检测miR-762及NF2 mRNA相对表达水平;Western blot实验检测Merlin、Yes相关蛋白1(YAP1)、p-YAP1、裂解半胱氨酸蛋白酶-3(C-caspase-3)、E钙粘附蛋白(E-cadherin)、波形蛋白(Vimentin)相对表达水平。结果TPC-1细胞存活率随ADVO作用浓度增大而降低(P<0.05),60μg/mL ADVO浓度作用下的细胞存活率接近50%;与空白组比较,ADVO组细胞存活率、miR-762及YAP1、Vimentin蛋白相对表达水平降低,细胞凋亡率、NF2 mRNA与Merlin、p-YAP1、C-caspase-3和E-cadherin蛋白相对表达水平升高、细胞迁移及侵袭数减少(P<0.05),miR-762 mimics组细胞存活率、miR-762及YAP1、Vimentin蛋白相对表达水平升高,细胞凋亡率、NF2 mRNA与Merlin、p-YAP1、C-caspase-3和E-cadherin蛋白相对表达水平降低、细胞迁移及侵袭数增加(P<0.05);ADVO可减弱miR-762过表达的作用效果,抑制TPC-1细胞恶性生物学行为(P<0.05);经生物信息学预测和双荧光素酶报告基因实验验证,NF2基因可能为miR-762的潜在靶基因。结论ADVO可抑制TPC-1细胞增殖、侵袭及迁移并促进其凋亡,其作用机制可能与抑制miR-762表达并提高其靶基因NF2编码Merlin蛋白水平,激活相关抑癌信号有关。

不同品种油橄榄果油脂品质差异形成的转录组学分析

为从基因水平研究不同品种油橄榄果油脂品质差异及其差异基因的代谢通路,以甘肃省陇南市两个油橄榄品种‘城固-32’和‘奇迹’第4成熟度果实为材料,分析两个品种油橄榄果油脂组成成分与理化性质差异,在此基础上,运用RNA-seq技术进行转录组分析,确定不同品种油橄榄果油脂品质形成差异的关键表达基因和相关代谢通路。结果表明:‘城固-32’和‘奇迹’油脂脂肪酸组成及含量、总酚和总黄酮含量、酸值和过氧化值有明显差异,在品质上表现为‘奇迹’优于‘城固-32’;两个品种油橄榄共筛选出10114条差异基因,这些差异基因与核苷酸合成、代谢和生物调节相关的通路有关,主要在代谢途径、次生代谢产物生物合成途径中富集;差异基因在光合作用、碳固定代谢、谷胱甘肽代谢、细胞色素P450代谢通路中的上调或下调对油橄榄果油脂差异的形成具有重要作用。

添加植物油降低饲粮n-6/n-3多不饱和脂肪酸比值对生长肥育猪脂肪沉积和脂肪酸组成及脂代谢相关基因表达的影响

本试验旨在研究饲粮添加植物油降低n-6/n-3多不饱和脂肪酸(PUFA)比值对生长肥育猪脂肪沉积和脂肪酸组成及脂代谢相关基因表达的影响。试验选用54头体重[(45.30±1.72)kg]相近的“杜×长×大”三元杂交猪,随机分为3组,每组6个重复,每个重复3头。对照组饲喂基础饲粮,试验组分别饲喂含4.5%菜籽油(菜籽油组)和4.5%混合油(混合油组,菜籽油∶亚麻籽油=1∶1)的饲粮。3组饲粮粗脂肪含量分别为2.76%、7.24%和7.24%,n-6/n-3 PUFA比值分别约为10∶1、4∶1和2∶1,消化能和粗蛋白质含量基本相同。预试期7 d,正试期42 d。结果表明:1)与对照组相比,饲粮添加植物油显著提高生长肥育猪背最长肌肌内脂肪(IMF)含量(P<0.05),对平均日增重(ADG)、平均日采食量(ADFI)、料重比(F/G)及背膘厚无显著影响(P>0.05)。2)与对照组相比,饲粮添加植物油显著提高背最长肌和皮下脂肪n-3及总PUFA含量(P<0.05),降低n-6/n-3 PUFA比值(P<0.05);与菜籽油组相比,混合油组背最长肌和皮下脂肪n-3 PUFA含量显著提高(P<0.05),n-6/n-3 PUFA比值显著降低(P<0.05)。3)与对照组相比,饲粮添加植物油显著提高背最长肌和皮下脂肪组织脂肪酸转位酶/CD36(FAT/CD36)和脂肪酸转运蛋白1(FATP1)mRNA相对表达量(P<0.05),且混合油组背最长肌FAT/CD36和脂肪酸转运蛋白4(FATP4)mRNA相对表达量显著高于菜籽油组(P<0.05)。4)与对照组相比,饲粮添加植物油显著提高背最长肌碳水化合物反应元件结合蛋白(ChREBP)、葡萄糖转运子4(Glut4)及脂肪酸合酶(FAS)、乙酰辅酶A羧化酶(ACC)、脂肪酸延伸酶5(Elovl5)和脂肪酸延伸酶6(Elovl6)等生脂基因mRNA相对表达量(P<0.05),显著降低皮下脂肪组织过氧化物酶体增殖物激活受体γ(PPARγ)、维甲酸X受体α(RXRα)及生脂基因mRNA相对表达量(P<0.05)。综上所述,添加植物油降低饲粮n-6/n-3 PUFA比值可上调FAT/CD36和FATP1表达,促进猪肌肉和皮下脂肪组织吸收和利用饲粮长链脂肪酸合成脂肪;下调PPARγ及生脂基因表达,抑制皮下脂肪组织从头脂肪合成,而上调ChREBP及生脂基因表达,促进背最长肌脂肪合成,提高IMF含量,且饲粮n-6/n-3 PUFA比值为2∶1时能更有效地改善组织n-3 PUFA含量和n-6/n-3 PUFA比值。

沙棘油脂合成关键基因GPD1和DGAT的克隆及功能验证

【目的】沙棘是水土保持和防沙治沙的重要木本油料树种,种子油富含生物活性成分,种子含油率低是制约沙棘开发和利用的主要瓶颈。本研究通过克隆沙棘种子油脂合成关键基因GPD1(glycerol-3-phosphate dehydrogenase)、DGAT1(diacylglycerol acyltransferase 1)和DGAT2(diacylglycerol acyltransferase 2),并进行了拟南芥异源过表达,以期为高油沙棘培育提供科学依据。【方法】基于沙棘转录组测序结果,以沙棘种子cDNA为模板克隆HrGPD1、HrDGAT1和HrDGAT2基因,通过In-fusion连接技术构建pCAMBIA1300-mCherry表达载体,通过农杆菌介导法将表达载体转入农杆菌GV3101,采用农杆菌蘸花法获取过表达油脂合成关键基因的拟南芥植株。利用实时荧光定量qRT-PCR分析HrGPD1、HrDGAT1和HrDGAT2在转基因拟南芥中的表达情况,利用氯仿甲醇法对T2代转基因和野生型的拟南芥进行含油量检测。【结果】通过PCR技术扩增得到了正确的HrGPD1、HrDGAT1和HrDGAT2基因序列,HrGPD1全长975 bp,编码氨基酸324个,分子量为35.55 kDa,等电点为5.36;HrDGAT1全长1608 bp,编码氨基酸535个,分子量为61.24 kDa,等电点为8.84;HrDGAT2全长993 bp,编码氨基酸330个,分子量为37.21 kDa,等电点为9.72。GPD1蛋白序列中含有1个保守结构域,属于甘油-3-磷酸脱氢酶家族成员,与枣和桑树亲缘关系较近;DGAT1、DGAT2蛋白序列中各含有1个保守结构域,属于膜结合O-酰基转移酶家族成员,DGAT1与枣亲缘关系较近,DGAT2与苹果和梨亲缘关系较近。通过农杆菌转化技术得到了稳定过表达HrGPD1、HrDGAT1和HrDGAT2的拟南芥株系。与野生型相比,过表达HrGPD1、HrDGAT1和HrDGAT2基因拟南芥T2代种子的含油量分别提高了5.09%、4.73%和2.51%。【结论】克隆获得沙棘HrGPD1、HrDGAT1和HrDGAT2的全长cDNA序列并鉴定功能,发现HrGPD1、HrDGAT1和HrDGAT2可以提高转基因拟南芥的种子含油量,为沙棘高油品种的培育提供了基因材料,对沙棘种子产油量提升具有重要意义。