铁十字秋海棠斑叶发育过程内参基因筛选及验证

doi:10.16420/j.issn.0513-353x.2021-0397

摘要/Abstract

摘要：

为研究铁十字秋海棠（Begonia masoniana）斑叶发育过程中最适内参基因与花青素苷合成相关基因的表达,以铁十字秋海棠7个时期的叶片为材料,通过实时荧光定量PCR技术、GeNorm、NormFinder和BestKeeper对11个候选内参基因（PP2A、EF1α、GADPH、CYP、elF4A、ACT2、18S、UBQ10、ACT7、PPR和HDH）进行表达稳定性评估,最适内参基因为ACT7和PP2A。分别以ACT7和PP2A为内参基因对6个花青素苷合成通路相关基因（CHS、F3H、F3’H、FLS、DFR和UFGT）的表达量进行分析,结果显示该通路相关基因表达先升后降,与叶片花青素苷含量变化趋势较为一致。

关键词: 铁十字秋海棠, 实时荧光定量PCR, 内参基因, 花青素苷合成基因, 表达分析

Abstract:

In order to research the suitable reference genes and the expression of the anthocyanin biosynthesis genes in the leaves of Begonia masoniana at different developmental stages,the expression stability of 11 genes（PP2A,EF1α,GADPH,CYP,elF4A,ACT2,18S,UBQ10,ACT7,PPR and HDH） was evaluated by real-time PCR,GeNorm,NormFinder and BestKeeper. The results showed that ACT7 and PP2A can be used as the suitable reference genes for gene expression analysis in B. masoniana. The expression levels of key genes involved in anthocyanin biosynthesis such as CHS,F3H,F3’H,FLS,DFR and UFGT were measured in leaves using the selected reference genes. The expression of the six anthocyanin pathway genes was higher in younger leaves and then declined during the later development stages,which was consistent with the change in the anthocyanin content in variegated leaves.

Key words: Begonia masoniana, quantitative real-time PCR, reference gene, anthocyanin biosynthesis genes, expression analysis

中图分类号:

S682

杨婷, 薛珍珍, 李娜, 郎校安, 李凌飞, 钟春梅. 铁十字秋海棠斑叶发育过程内参基因筛选及验证[J]. 园艺学报, 2021, 48(11): 2251-2261.

YANG Ting, XUE Zhenzhen, LI Na, LANG Xiaoan, LI Lingfei, ZHONG Chunmei. Reference Genes Selection and Validation in Begonia masoniana Leaves of Different Developmental Stages[J]. Acta Horticulturae Sinica, 2021, 48(11): 2251-2261.

图/表 12

参考文献 33

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时期 Stage	叶片长/cm Leaf length	叶片宽/cm Leaf width	生长天数/d Growth days	特征描述 Feature description
S1	1.2 ~ 1.6	1.0 ~ 1.2	2 ~ 3	叶片卷曲,表皮毛较长,全叶呈红色 Leaf curly,hirsute-villous,whole leaf is red
S2	2.2 ~ 2.8	1.8 ~ 2.2	6 ~ 8	叶片舒张,表皮毛长,叶片质地较厚,全叶呈红色 Leaf expansion,hirsute-villous,thick,whole leaf is red
S3	4.1 ~ 5.1	3.1 ~ 3.7	10 ~ 12	叶斑初显,沿主叶脉规则分布,全叶呈红色 Leaf variegation appearing,distributed along the main vein,whole leaf is red
S4	6.8 ~ 7.4	4.6 ~ 6.2	15 ~ 18	叶斑颜色继续加深,叶斑以外部分叶色偏黄 Leaf variegation deepen,other parts of leaf show yellowish
S5	10.2 ~ 12.8	7.7 ~ 8.7	23 ~ 25	叶斑明显,叶斑以外部分叶色逐渐转绿 Leaf variegation begin obvious,other parts of leaf turn green
S6	12.7 ~ 15.0	9.2 ~ 11.9	33 ~ 35	叶片继续生长,叶斑以外部分叶色全绿 Leaf continues to grow,and other parts except leaf variegation are all green
S7	20.1 ~ 21.5	15.2 ~ 16.1	48 ~ 52	叶片大小达到最大 Leaf size reaches maximum

时期 Stage	叶片长/cm Leaf length	叶片宽/cm Leaf width	生长天数/d Growth days	特征描述 Feature description
S1	1.2 ~ 1.6	1.0 ~ 1.2	2 ~ 3	叶片卷曲,表皮毛较长,全叶呈红色 Leaf curly,hirsute-villous,whole leaf is red
S2	2.2 ~ 2.8	1.8 ~ 2.2	6 ~ 8	叶片舒张,表皮毛长,叶片质地较厚,全叶呈红色 Leaf expansion,hirsute-villous,thick,whole leaf is red
S3	4.1 ~ 5.1	3.1 ~ 3.7	10 ~ 12	叶斑初显,沿主叶脉规则分布,全叶呈红色 Leaf variegation appearing,distributed along the main vein,whole leaf is red
S4	6.8 ~ 7.4	4.6 ~ 6.2	15 ~ 18	叶斑颜色继续加深,叶斑以外部分叶色偏黄 Leaf variegation deepen,other parts of leaf show yellowish
S5	10.2 ~ 12.8	7.7 ~ 8.7	23 ~ 25	叶斑明显,叶斑以外部分叶色逐渐转绿 Leaf variegation begin obvious,other parts of leaf turn green
S6	12.7 ~ 15.0	9.2 ~ 11.9	33 ~ 35	叶片继续生长,叶斑以外部分叶色全绿 Leaf continues to grow,and other parts except leaf variegation are all green
S7	20.1 ~ 21.5	15.2 ~ 16.1	48 ~ 52	叶片大小达到最大 Leaf size reaches maximum

基因 Gene	引物序列（5′-3′） Primer sequence
CHS	F:TGCACCACAAGCGGAGTCGA;R:AGCAAGATCCTTGGCGACCCT
F3H	F:TTCCAGAACCCAGCGCCAGA;R:TCAGCCTGGCAAGCTCAAGGT
F3’H	F:GCGACTTCATTCCGGCGCTT;R:ACCGCCGATCCGCTTGTGTT
FLS	F:TGAGCAGCCCGGAATCACCA;R:TGTCGCACACCACACGTTCCT
DFR	F:AGACATTGGCAGAGCAGGCG;R:AGTGATCAGGCTCGGTGGCA
UFGT	F:TGAATGCCGCCCCAGAAAGCT;R:CGGCGAACCACAAGAAAGCGT

基因 Gene	引物序列（5′-3′） Primer sequence
CHS	F:TGCACCACAAGCGGAGTCGA;R:AGCAAGATCCTTGGCGACCCT
F3H	F:TTCCAGAACCCAGCGCCAGA;R:TCAGCCTGGCAAGCTCAAGGT
F3’H	F:GCGACTTCATTCCGGCGCTT;R:ACCGCCGATCCGCTTGTGTT
FLS	F:TGAGCAGCCCGGAATCACCA;R:TGTCGCACACCACACGTTCCT
DFR	F:AGACATTGGCAGAGCAGGCG;R:AGTGATCAGGCTCGGTGGCA
UFGT	F:TGAATGCCGCCCCAGAAAGCT;R:CGGCGAACCACAAGAAAGCGT

基因 Gene	基因名称 Gene name	引物序列（5′-3′） Primer sequence	斜率 k	扩增效率/% E	相关系数 R²
PP2A	Serine/threonine-PP2A catalytic subunit	F:TCCTGATGGAGTGCAAGCCGT R:ACAGACTGTCACCGGGCACT	-3.31	100.50	0.9984
EF1α	Elongation factor 1α	F:TGCAGCTTGGCTCGGCAGAT R:TGGCAGGGGCGTGATCTAACA	-3.41	96.45	0.9996
GAPDH	Glyceraldehyde-3-phosphate dehydrogenase	F:TGACGTTGTGGCAGCTCAGGT R:TCCGGCCAATGCGTCCGAAT	-3.32	100.08	0.9961
CYP	Cyclophilin	F:ACGGTGCCAAGTTTGCCGAC R:AACTGTGAGCCGTTGGTTCCG	-3.34	99.25	0.9988
elF4A	Eukaryotic translation initiation factor 4A-1	F:ACCTCCATCGAATCGGCCGT R:TGCTGGCAACTCCTCCACCA	-3.20	105.35	0.9995
ACT2	Actin 2	F:TTGCTCACAGAGGCACCGCT R:ACCGGTAGTACGACCACTGGCA	-3.33	99.66	0.9935
18S	18S Ribosomal RNA	F:TGACGGATCGCACGGCCTTT R:TTCTCCGTCACCCGTCACCA	-3.45	94.92	0.9966
UBQ10	Polyubiquitin 10	F:ACGCTTCGAGGTAGGTTTCTTGT R:ACGCATCGAAAACAACAACCGCA	-3.50	93.07	0.9991
ACT7	Actin 7	F:TCGTGCTTGGAGGTTCGGCT R:CCAGCCTTCACCATTCCAGTTCCA	-3.27	102.21	0.9964
PPR	Pentatricopeptide repeat superfamily protein	F:AGAAGTGCGAGTGGCTACCT R:CACAAACCCGCCGATCAGAGT	-3.19	105.82	0.9996
HDH	Histidinol dehydrogenase	F:ATGTCCCTGGGGGTACTGCTGT R:TGCCATCCTGACTTGGGGGA	-3.36	98.44	0.9997