Effect Comparison of RNA Extraction and Virus Detection of Different Parts of Four Organs of Apple

doi:10.7668/hbnxb.20193519

Abstract

Abstract:

In order to explore the effect of RNA extraction and virus detection of different parts of apple organs,apple plants infected by three viruses were used as experimental materials.Adsorption column method was used to extract total RNA from flowers(pistil,anther,filament,petals,calyx,receptacle,and flower stalk),leaves(petiole,mesophyll,and veins),fruits(fruit stalk,peel,flesh,score,episperm,and endosperm)and branches(phloem),a total of 17 tissues of four organs.The extraction effects of different sampling parts were compared,the detection effects of three viruses in different parts of organs were analyzed by regular RT-PCR,and the relative content of viruses in different parts of organs were analyzed by Real-time fluorescence quantitative PCR.The results showed that the effects of RNA extraction from different organs of apple were different,with the highest concentration in leaves,followed by flowers and phloem of branches,and the lowest concentration in fruits.Anther,filament and petiole were best extracted from flowers and endosperm and petiole were best extracted from fruits and leaves,respectively.Three apple viruses could be detected in 16 tissues except endosperm.Quantitative analysis showed that the relative content of the three apple viruses was the highest in flowers.Furthermore,the relative contents of viruses in pistil,episperm and petiole were correspondingly higher than that in other tissue parts of flower,fruit and leaf,respectively.In addition,the content of the three viruses in different tissue parts of fruits and leaves showed similar trends,but there were certain differences in the distribution of different tissue parts of flowers.

Key words: Apple, RNA extraction, Virus detection, Comparison of viral concentration

JIA Xiaojun, DONG Yafeng, ZHANG Zunping, FAN Xudong, REN Fang, ZHANG Baodong, HU Guojun. Effect Comparison of RNA Extraction and Virus Detection of Different Parts of Four Organs of Apple[J]. Acta Agriculturae Boreali-Sinica, 2022, 37(S1): 283-290. doi: 10.7668/hbnxb.20193519.

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Figures/Tables 11

References 30

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病毒 Viruses	引物 Primers	序列(5'-3') Sequences (5'-3')	片段大小/bp Products size	参考 References
常规RT-PCR Regular RT-PCR
ASGV	ASGV-F	CTGCAAGACCGCGACCAAGTTT	524	[14]
	ASGV-R	CCCGCTGTTGGATTTGATACACCTC
ACLSV	ACLSV-F	ATCGCAGAAGGGGATATTCCAAT	1 016	[15]
	ACLSV-R	TGTCKAGCAAACTGAAAYTCTCT
ASPV	ASPV-F	AGCGGTTGCCTATTTTTGCTCC	291	[16]
	ASPV-R	GTGAGGTCAAAGATGCTGAAACC
荧光定量RT-PCR Real-time RT-PCR
ASGV	GV-F2	GCAGAACTCTTTGAACGAATG	124	[17]
	GV-R2	GGGCCATTTCTTGTAAATGTTGGTG
ACLSV	CL-F2	CCTGAGGCAAGAAACGGGTTGGT	113
	CL-R2	TTGAAGTCGAACATCAGCTCCGGGTA
ASPV	PV-F1	TCAAGCAGACTGAGGGGTGCACTT	88
	PV-R1	GGACTTTGAGTTTGCAGCAT
内参	Actin-F	GGATTTGCTGGTGATGATGCT	178	[18]
Internal control	Actin-R	AGTTGCTCACTATGCCGTGCT

病毒 Viruses	引物 Primers	序列(5'-3') Sequences (5'-3')	片段大小/bp Products size	参考 References
常规RT-PCR Regular RT-PCR
ASGV	ASGV-F	CTGCAAGACCGCGACCAAGTTT	524	[14]
	ASGV-R	CCCGCTGTTGGATTTGATACACCTC
ACLSV	ACLSV-F	ATCGCAGAAGGGGATATTCCAAT	1 016	[15]
	ACLSV-R	TGTCKAGCAAACTGAAAYTCTCT
ASPV	ASPV-F	AGCGGTTGCCTATTTTTGCTCC	291	[16]
	ASPV-R	GTGAGGTCAAAGATGCTGAAACC
荧光定量RT-PCR Real-time RT-PCR
ASGV	GV-F2	GCAGAACTCTTTGAACGAATG	124	[17]
	GV-R2	GGGCCATTTCTTGTAAATGTTGGTG
ACLSV	CL-F2	CCTGAGGCAAGAAACGGGTTGGT	113
	CL-R2	TTGAAGTCGAACATCAGCTCCGGGTA
ASPV	PV-F1	TCAAGCAGACTGAGGGGTGCACTT	88
	PV-R1	GGACTTTGAGTTTGCAGCAT
内参	Actin-F	GGATTTGCTGGTGATGATGCT	178	[18]
Internal control	Actin-R	AGTTGCTCACTATGCCGTGCT

取样部位 Sampling parts	浓度/(ng/μL)Concentration			A_260/280
取样部位 Sampling parts	富士 Fuji	锦绣海棠 Jinxiuhaitang	信浓黄 Xinnonghuang	富士 Fuji	锦绣海棠 Jinxiuhaitang	信浓黄 Xinnonghuang
叶片Leaves	706.4±68.7a	633.9±75.7a	374.9±40.6a	2.08	2.09	2.08
花Flowers	293.0±48.6b	580.0±67.7ab	296.5±51.3ab	2.09	2.08	2.08
枝条Branches	243.4±12.6bc	307.2±102.2bc	169.3±35.0bc	2.06	2.07	2.07
果实Fruits	66.1±4.7c	89.6±5.1c	125.4±4.5c	2.07	2.07	2.09

取样部位 Sampling parts	浓度/(ng/μL)Concentration			A_260/280
取样部位 Sampling parts	富士 Fuji	锦绣海棠 Jinxiuhaitang	信浓黄 Xinnonghuang	富士 Fuji	锦绣海棠 Jinxiuhaitang	信浓黄 Xinnonghuang
叶片Leaves	706.4±68.7a	633.9±75.7a	374.9±40.6a	2.08	2.09	2.08
花Flowers	293.0±48.6b	580.0±67.7ab	296.5±51.3ab	2.09	2.08	2.08
枝条Branches	243.4±12.6bc	307.2±102.2bc	169.3±35.0bc	2.06	2.07	2.07
果实Fruits	66.1±4.7c	89.6±5.1c	125.4±4.5c	2.07	2.07	2.09

取样部位 Sampling parts	浓度/(ng/μL)Concentration
取样部位 Sampling parts	富士 Fuji	锦绣海棠 Jinxiuhaitang	清明 Qingming	华红 Huahong	富士 Fuji	锦绣海棠 Jinxiuhaitang	清明 Qingming	华红 Huahong
雌蕊Pistil	353.7±59.4bc	394.2±38.4bc	425.5±20.7bcd	344.3±83.3cd	2.09	2.06	2.09	2.08
花药Anther	1 112.1±148.8ab	1 000.6±87.2a	1 256.1±138.8a	999.3±52.2ab	2.10	2.08	2.09	2.07
花丝Filament	836.9±166.1abc	541.2±176.2abc	755.2±77.8abc	1 018.5±171.7a	2.06	2.06	2.09	2.10
花瓣Petal	347.3±20.0bc	346.3±48.4bc	157.2±14.6d	221.1±37.7d	2.05	2.09	2.08	2.09
花萼Calyx	154.8±12.8c	370.3±7.5bc	316.0±35.0cd	465.7±121.1bcd	2.06	2.09	2.10	2.10
花托Receptacle	432.7±44.5bc	102.1±45.4c	346.6±84.3cd	436.0±111.5cd	2.09	2.07	2.07	2.10
花柄Flower stalk	1 479.9±344.1a	695.5±68.3ab	926.2±200.4ab	877.9±8.1abc	2.09	2.05	2.08	2.08

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