Scientia Agricultura Sinica ›› 2019, Vol. 52 ›› Issue (13): 2341-2351.doi: 10.3864/j.issn.0578-1752.2019.13.012

• ANIMAL SCIENCE·VETERINARY SCIENCE·RESOURCE INSECT • Previous Articles     Next Articles

Expression Profile and Correlations of Kruppel Like Factors During Caprine (Capra Hircus) Preadipocyte Differentiation

ZHU JiangJiang1,LIN YaQiu1(),WANG Yong1,2,LIN Sen2   

  1. 1 Qinghai-Tibetan Plateau Animal Genetic Resource Reservation and Utilization Key Laboratory of Sichuan Province, Chengdu 610041
    2 Key Laboratory of Qinghai-Tibetan Plateau Animal Genetic Resource Reservation and Utilization (Southwest Minzu University), Ministry of Education, Chengdu 610041
  • Received:2018-01-24 Accepted:2019-05-10 Online:2019-07-01 Published:2019-07-11
  • Contact: YaQiu LIN E-mail:linyq1999@163.com

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Abstract:

【Objective】 The aim of this study was to optimize the culture system of goat preadipocytes in vitro and elucidate the expression patterns of Kruppel like factors (KLFs) during preadipocytes differentiation. 【Method】 Three 7-day-old Jianzhou Big-Eared goats were selected randomly from Sichuan Jianyang DAGEDA Animal Husbandry Co. Ltd. as experimental animal. After bloodletting death of goats, their subcutaneous fat tissues were collected in a sterile room. The goat subcutaneous preadipocytes were collected using collagenase I digestion method. The cells at Generation Three were then passaged to a 6-well plate when the cells were grown to 80% density. The combined use of were used for preadipocytes differentiate inducing. The cells were then collected after cultivation for 0 d, 3 d, 5 d and 7 d. The total RNA was extracted by using Trizol method. Real-time fluorescent quantitative PCR (RT-qPCR) was performed to determine the expression levels of 11 members of KLFs, including KLF2-10, KLF12 and KLF15, during goat preadipocytes differentiation. Pearson correlation coefficient was used for expression correlation analysis between KLF individuals. 【Result】 The results showed that the combined use of 150 μmol·L -1oleic acid and 10 mg·L -1 insulin promoted the formation of small lipid droplets after 24 h cultivation. After 48 h, the lipid droplets became bigger and much more. The differentiation of the preadipocytes were enhanced significantly after 120 h comparing with preadipocytes at 0 h. The results of RT-qPCR showed that the combined use of oleic acid and insulin stimulated the expression of peroxisome proliferator-activated receptor-gamma (PPARγ) continuously companying the differentiation of preadipocytes, and toped at 7 d which significantly higher than other groups (P＜0.01). The expression levels of KLF2, KLF3, KLF4, KLF6, KLF7 and KLF15 in adipocytes were significantly (P＜0.05) or extremely significantly (P＜0.01) higher than those in preadipocytes. While the expression of KLF3, KLF6 and KLF15 were topped at 5 d, the KLF2 and KLF4 toped at 7 d. However, the KLF7 expressed most at 3 d. For the other KLF individuals, the expression levels of KLF5, KLF8, KLF9, KLF10 and KLF12 in preadipocytes were extremely significantly higher (P＜0.01) than those in adipocytes. The expression of KLF8, KLF10 and KLF12 bottomed at 5 d, while that of the KLF5 and KLF9 bottomed at 7 d. Correlation analysis showed that KLF individual significantly (P＜0.05) correlated with each other across cell differentiation. KLF12 was strongly correlated with 6 individuals, followed by KLF4 and KLF9 both of which were significantly correlated with 5 individuals. However, KLF2 was not significantly correlated with other individuals. 【Conclusion】 In this study, the expression patterns of KLFs during goat preadipocytes differentiation were clarified, and also the correlations among KLF individuals. These results provided important basic data for elucidating the molecular mechanism of goat preadipocytes differentiation.

Key words: goat, preadipocytes, differentiation, Kruppel like factors

Table 1

Primers information"

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

Fig. 1

Induced differentiation of goat preadipocytes (100×) A. 24 h after cell differentiation inducing; B. 48 h after cell differentiation inducing; C. 72 h after cell differentiation inducing; D. 120 h after cell differentiation inducing"

Fig. 2

The expression of PPARγ during preadipocyte differentiation Different capital letters represents P<0.01, different lower-case letters represents P<0.05. The same as below "

Fig. 3

The expression of KLF2, KLF3, KLF4, KLF6, KLF7 and KLF15 during preadipocyte differentiation Different capital letters represents P<0.01, different lower-case letters represents P<0.05"

Fig. 4

The expression of KLF5, KLF8, KLF9, KLF10 and KLF12 during preadipocyte differentiation Different capital letters represents P<0.01, different lower-case letters represents P<0.05"

Table 2

The expression correlations of KLF in caprine (Capra hircus) preadipocyte differentiation"

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

Fig. 5

The correlation network among KLFs "

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