Selection of Reference Genes for Normalization of Quantitative Real-time PCR Analysis in Secondary Laticifer Differentiation Induced by Trichostatin A in Rubber Tree (Hevea brasiliensis Muell. Arg.)

doi:10.3969/j.issn.1000-2561.2020.03.012

Abstract

Abstract:

The identification of differentially expressed genes associated with laticifer differentiation is crucial for understanding the molecular mechanism of laticifer differentiation. The selection of a suitable reference gene is a precondition for accurate gene expression analysis by quantitative real-time PCR (qRT-PCR). In the present study, the expression stability of 22 candidate reference genes were evaluated on the basis of TSA-induced secondary laticifer differentiation using geNorm and NormFinder algorithms. According to the analysis of geNorm and NormFinder, UBC3, UBC4, eIF1Aa were the top three stable genes and ROC3, PTP, CYP2 were the last three stable genes in the process of TSA-induced secondary laticifer differentiation. The expression patterns of HDA1 and HDA2 in TSA treated bark were analyzed based on UBC3, Actin and ROC3 as the reference gene respectively. The results showed that UBC3 could serve as a qRT-PCR reference gene to analyze the gene expression pattern in TSA-induced secondary laticifer differentiation. And ROC3 was not suitable as reference genes for qRT-PCR.

Key words: Hevea brasiliensis Muell. Arg., laticifer differentiation, trichostatin A, reference genes

CLC Number:

S794.1

WU Shaohua,ZHANG Shixin,YANG Shuguang,TIAN Weimin. Selection of Reference Genes for Normalization of Quantitative Real-time PCR Analysis in Secondary Laticifer Differentiation Induced by Trichostatin A in Rubber Tree (Hevea brasiliensis Muell. Arg.)[J]. Chinese Journal of Tropical Crops, 2020, 41(3): 504-512.

Figures/Tables 7

References 24

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基因 Gene	上游引物 Forward primer sequence (5'-3')	下游引物 Reverse primer sequence (5'-3')	扩增长度 Amplicon length/bp
18S	GCTCGAAGACGATCAGATACC	TTCAGCCTTGCGACCATAC	146
Actin	GATTCCGTTGCCCAGAAGTC	CACCACTCAGCACAATGTTACC	144
ADF	GGAGAAGGCAAAGCAAGTC	GCATATCGGCACTCATCAG	99
ADF4	GTGCCGATATGCTGTCTATGATTT	TCCCTCTTGAACCTGTCCTTG	147
CYP2	CTGTACTGCCAAGACTGAG	CTATGGCCTTCACCACATC	86
eIF1Aa	GCGTGACTATCAGGACGACAA	CAAGACCTCCAGCAATACCCT	128
eIF1Ab	TGGTGTTGGTGTGGTGATAG	TATTCCTGCCCATCTTCCTTG	135
eIF2	CGACCTTTGATCCGTTTGCT	CTTCCTACCATTCCGTTGCT	98
eIF3	CGAAACCCAGATTCCTTCTACCT	CTGCACAGTAGTCAAGCTCTTTC	133
FP	ATCAGCTTGACGCAGGTTAC	GAAACAGACGCTTCTGAGTG	99
PTP	TGCCCTCCAATTCAACTG	TGCACCACAACGACATTC	86
RH2a	CCATCACCCAGGCTGTTATTTTC	GTAAAGTTATTGCTCCGCATCTTC	82
RH2b	AGGTGGATTGGCTAACTGAG	GAGCCCAAACATCAGTAGTG	150
RH8	TCACAGGGTTGGTAGATCAG	CCAAGCTCTTGCTCAATCC	105
ROC3	ATGGAGCTATTCGCGGATAC	GGTGGAAACTCGATCCTTTG	121
TCPB	CAGACAGTCAATGACAGTAGGGTT	ATCCCTGCAGTGCAATCCTCC	239
UBC1	ACTTTGCCCTTGATGCCT	GCGTTAGTTGGGATCTGGT	102
UBC2a	CATTTATGCGGATGGAAGCA	CAGGGGAGTTTGGATTTGGA	125
UBC2b	CGACCAAGTTTTCATTTCGGGTG	AGTCTCTTCTTTGCTGGGGTTG	95
UBC3	CCCTGATGATCCACTTTCTG	ACCATCATGCACCACTTG	113
UBC4	TCCTTATGAGGGCGGAGTC	CAAGAACCGCACTTGAGGAG	82
YLS8	CCTCGTCGTCATCCGATTC	CAGGCACCTCAGTGATGTC	131
HDA1	CTATTGGGTGGTGGTGGTTAT	CATGCTGTGGCATCTTGTTATC	106
HDA2	TGGGCAAGGGAAGAACTATG	TCTGGATGATGGGACGAAAC	92

基因 Gene	上游引物 Forward primer sequence (5'-3')	下游引物 Reverse primer sequence (5'-3')	扩增长度 Amplicon length/bp
18S	GCTCGAAGACGATCAGATACC	TTCAGCCTTGCGACCATAC	146
Actin	GATTCCGTTGCCCAGAAGTC	CACCACTCAGCACAATGTTACC	144
ADF	GGAGAAGGCAAAGCAAGTC	GCATATCGGCACTCATCAG	99
ADF4	GTGCCGATATGCTGTCTATGATTT	TCCCTCTTGAACCTGTCCTTG	147
CYP2	CTGTACTGCCAAGACTGAG	CTATGGCCTTCACCACATC	86
eIF1Aa	GCGTGACTATCAGGACGACAA	CAAGACCTCCAGCAATACCCT	128
eIF1Ab	TGGTGTTGGTGTGGTGATAG	TATTCCTGCCCATCTTCCTTG	135
eIF2	CGACCTTTGATCCGTTTGCT	CTTCCTACCATTCCGTTGCT	98
eIF3	CGAAACCCAGATTCCTTCTACCT	CTGCACAGTAGTCAAGCTCTTTC	133
FP	ATCAGCTTGACGCAGGTTAC	GAAACAGACGCTTCTGAGTG	99
PTP	TGCCCTCCAATTCAACTG	TGCACCACAACGACATTC	86
RH2a	CCATCACCCAGGCTGTTATTTTC	GTAAAGTTATTGCTCCGCATCTTC	82
RH2b	AGGTGGATTGGCTAACTGAG	GAGCCCAAACATCAGTAGTG	150
RH8	TCACAGGGTTGGTAGATCAG	CCAAGCTCTTGCTCAATCC	105
ROC3	ATGGAGCTATTCGCGGATAC	GGTGGAAACTCGATCCTTTG	121
TCPB	CAGACAGTCAATGACAGTAGGGTT	ATCCCTGCAGTGCAATCCTCC	239
UBC1	ACTTTGCCCTTGATGCCT	GCGTTAGTTGGGATCTGGT	102
UBC2a	CATTTATGCGGATGGAAGCA	CAGGGGAGTTTGGATTTGGA	125
UBC2b	CGACCAAGTTTTCATTTCGGGTG	AGTCTCTTCTTTGCTGGGGTTG	95
UBC3	CCCTGATGATCCACTTTCTG	ACCATCATGCACCACTTG	113
UBC4	TCCTTATGAGGGCGGAGTC	CAAGAACCGCACTTGAGGAG	82
YLS8	CCTCGTCGTCATCCGATTC	CAGGCACCTCAGTGATGTC	131
HDA1	CTATTGGGTGGTGGTGGTTAT	CATGCTGTGGCATCTTGTTATC	106
HDA2	TGGGCAAGGGAAGAACTATG	TCTGGATGATGGGACGAAAC	92