中国农业科学 ›› 2019, Vol. 52 ›› Issue (13): 2341-2351.doi: 10.3864/j.issn.0578-1752.2019.13.012
收稿日期:
2018-01-24
接受日期:
2019-05-10
出版日期:
2019-07-01
发布日期:
2019-07-11
通讯作者:
林亚秋
作者简介:
朱江江,E-mail:zhujiang4656@hotmail.com
基金资助:
ZHU JiangJiang1,LIN YaQiu1(),WANG Yong1,2,LIN Sen2
Received:
2018-01-24
Accepted:
2019-05-10
Online:
2019-07-01
Published:
2019-07-11
Contact:
YaQiu LIN
摘要:
【目的】 优化山羊前体脂肪细胞体外培养体系,揭示Kruppel样转录因子家族(KLFs)成员在前体脂肪细胞分化过程中的表达模式。 【方法】 于四川省简阳大哥大牧业有限公司随机选取3只7日龄简州大耳羊为研究对象。放血致死并清洗后,在无菌环境中取其皮下脂肪组织。利用Ⅰ型胶原酶消化法获得其皮下前体脂肪细胞,待细胞长至细胞密度为80%时传代至6孔培养板中(F3代),以“150 μmol·L -1油酸+10 mg·L -1胰岛素”前体脂肪细胞进行成脂诱导,并在诱导分化后0 d、3 d、5 d、7 d收集细胞,Trizol法提取细胞总RNA,利用实时荧光定量PCR(RT-qPCR)检测KLF2-10, KLF 12和KLF15共11个KLF转录因子家族成员在不同分化阶段细胞中的表达水平,并利用皮尔逊系数分析各个成员mRNA总体表达水平之间的相关性。 【结果】 150 μmol·L -1油酸+10 mg·L -1胰岛素处理前体脂肪细胞24 h后细胞中开始出现小脂滴,48 h后脂滴数量增多、体积增大,诱导120 h后脂肪细胞分化程度显著增加。RT-qPCR结果显示,脂肪分化标志基因过氧化物酶体增殖物活化受体γ(PPARγ)基因随着诱导分化的进行其表达水平持续上升,其中在第7天时其表达水平极显著高于其他各组(P<0.01)。KLF2、KLF3、KLF4、KLF6、KLF7和KLF15在脂肪细胞中表达水平显著(P<0.05)或极显著(P<0.01)高于前体脂肪细胞中表达水平,其中KLF3、KLF6和KLF15在分化5 d时表达水平最高,KLF2和KLF4表达水平在7 d时最高,而KLF7则在3 d时表达水平最高;KLF5、KLF8、KLF9、KLF10和KLF12在前体脂肪细胞中的表达水平极显著高于分化后脂肪细胞中的表达水平(P<0.01),其中KLF8、KLF10和KLF12的表达水平均在5 d时达到最低,而KLF5和KLF9则在7 d时表达水平最低;相关性分析结果显示KLF家族各成员mRNA在山羊前体脂肪细胞分化过程中总体表达水平存在相关性,KLF12与6个基因具有较强的相关性,其次为KLF4和KLF9,分别与5个成员具有较强的相关性,而KLF2与其他基因均没有较强的相关性。 【结论】 明确了KLF家族在山羊前体脂肪细胞分化过程中的表达模式,解析了各个成员mRNA表达之间的相关性,为阐明山羊前体脂肪细胞分化的分子机制提供重要的基础数据。
朱江江,林亚秋,王永,林森. 山羊Kruppel样转录因子家族在前体脂肪细胞分化中的表达模式及相关性分析[J]. 中国农业科学, 2019, 52(13): 2341-2351.
ZHU JiangJiang,LIN YaQiu,WANG Yong,LIN Sen. Expression Profile and Correlations of Kruppel Like Factors During Caprine (Capra Hircus) Preadipocyte Differentiation[J]. Scientia Agricultura Sinica, 2019, 52(13): 2341-2351.
表1
引物信息"
目的基因 Gene | 登录号 Accession No. | 引物序列(5′-3′) Primer sequences | 退火温度 TM (℃) | 产物长度 Products length (bp) |
---|---|---|---|---|
KLF2 | KU041748 | S: GCGGCAAGACCTACACCAA | 60 | 144 |
A: TGTGCTTGCGGTAGTGGC | ||||
KLF3 | KU041753 | S: CTCGGTGTCATACCCGTCTAAT | 60 | 128 |
A: CCATCTGCATCCCGTGAGAC | ||||
KLF4 | KU041754 | S: GTCGGTCATCAGTGTTAGCAAAGG | 62 | 126 |
A: ACGGTGCACGAGGAGACAGTCT | ||||
KLF5 | KU041751 | S:CACCTCCATCCTATGCTGCTAC | 62 | 169 |
A:CAGCCTGGGTAATCGCAGTAGT | ||||
KLF6 | KU041749 | S:GCCTCTGAGATCAAATTCGACA | 62 | 189 |
A:GGAGGACTCGCTGCTCACAT | ||||
KLF7 | KU041750 | S:TTCGGTGAGGACTTGGACTGTT | 60 | 148 |
A:TGTCCCGAGAGAGCAGAATGTC | ||||
KLF8 | KX247671 | S:GACTACAGCAAGAACCAGCAGC | 62 | 153 |
A:CTCCTGTATGGATTCTGCGGT | ||||
KLF9 | KX247670 | S:GGATACCTGGAAGGATTACTGC | 60 | 172 |
A:GGAAGGACTCGACCCAGATT | ||||
KLF10 | KU643198 | S:GTCAGATACTGCCAAGCCTCAC | 62 | 133 |
A:GTTACACAGTTGGGCATCAGC | ||||
KLF12 | KX247669 | S:GGTCTCCAAACGTCCACAAGTA | 62 | 169 |
A:GAAACGGCTGTAGGGGATGTC | ||||
KLF15 | KU041752 | S:CTGACTCGCAAACCCTGTGT | 62 | 192 |
A:ACTCGGGGAAGCTGAAATG | ||||
GAPDH | XM_005680968 | S:GCAAGTTCCACGGCACAG | 62 | 118 |
A:TCAGCACCAGCATCACCC |
表2
KLF家族各成员间表达水平的相关性"
KLF基因 KLF gene | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 12 | 15 |
---|---|---|---|---|---|---|---|---|---|---|---|
2 | 1 | 0.126 | 0.517 | -0.279 | 0.236 | 0.327 | -0.569 | -0.662 | -0.076 | -0.459 | -0.400 |
3 | 1 | 0.423 | -0.206 | 0.874** | 0.027 | -0.734* | -0.516 | -0.500 | -0.400 | 0.467 | |
4 | 1 | -0.841** | 0.299 | 0.770 | -0.853** | -0.890** | -0.840** | -0.984** | 0.316 | ||
5 | 1 | 0.078 | -0.617 | 0.595 | 0.734* | 0.636 | 0.833* | -0.085 | |||
6 | 1 | 0.029 | -0.648 | -0.436 | -0.438 | -0.296 | 0.534 | ||||
7 | 1 | -0.402 | -0.458 | -0.698 | -0.841** | 0.397 | |||||
8 | 1 | 0.959**# | 0.732* | 0.770# | -0.799# | ||||||
9 | 1 | 0.861*# | 0.842** | -0.820*# | |||||||
10 | 1 | 0.980**# | -0.982**# | ||||||||
12 | 1 | -0.988**# | |||||||||
15 | 1 |
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