In Situ Hybridization Analysis of Distinctive Ribonucleotide Reductase β Subunit Gene from Candidatus Liberibacter Asiaticus of Citrus

doi:10.16420/j.issn.0513-353x.2022-1095

Acta Horticulturae Sinica ›› 2023, Vol. 50 ›› Issue (12): 2689-2700.doi: 10.16420/j.issn.0513-353x.2022-1095

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In Situ Hybridization Analysis of Distinctive Ribonucleotide Reductase β Subunit Gene from Candidatus Liberibacter Asiaticus of Citrus

ZHANG Shaoran¹, ZONG Qi¹, LIU Yang², JIANG Ling²^,^*()

¹ National Key Laboratory of Agricultural Microbiology，Huazhong Agricultural University，Wuhan 430070，China
² National Key Laboratory of Germplasm Innovation and Utilization of Fruit and Vegetable Horticultural Crops，National Fruit Free-virus Germplasm Resource Indoor Conservation Center，Department of Horticulture and Forestry，Huazhong Agricultural University，Wuhan 430070，China

Received:2023-08-24 Revised:2023-11-13 Online:2023-12-25 Published:2023-12-29
Contact: JIANG Ling

Abstract

Abstract:

Candidatus Liberibacter asiaticus（CLas），can be determined by qPCR detection. However，the selection of different tissue samples will affect the results of qPCR detection. Therefore，it is necessary to study the location of CLas in citrus tissues. Based on the specific gene sequence of ribonucleotide reductase（RNR）β subunit of CLas of citrus Huanglongbing disease（HLB），123 bp antisense RNA probe and sense RNA probe was designed and prepared. In situ hybridization analysis was performed on the pathogen gene of CLas in shoot tip，leaf vein，petiole，clade and root tip，meanwhile，the pathogens were detected by qPCR. The results showed that the visual blue hybridization signals in sieve tube and companion cells of phloem in CLas-infected plants could be clearly observed under the microscope and the distribution of pathogens in the different organs was significantly different，compared with root tip and shoot-tip，RNR gene distribution in phloem of bark，petiole and vein was more accurate locating. The HLB-infected condition detected by qPCR and the visualization results of in situ hybridization shared the consistency in citrus nursery plants.

Key words: citrus, Candidatus Liberibacter asiaticus, RNA in situ hybridization, ribonucleotide reductase β subunit gene

ZHANG Shaoran, ZONG Qi, LIU Yang, JIANG Ling. In Situ Hybridization Analysis of Distinctive Ribonucleotide Reductase β Subunit Gene from Candidatus Liberibacter Asiaticus of Citrus[J]. Acta Horticulturae Sinica, 2023, 50(12): 2689-2700.

Figures/Tables 10

Table 1 Dehydration procedures of the samples for FAA fixed mL

程序 Procedure	蒸馏水 Distilled water	95%乙醇 95%Ethanol	叔丁醇 Tert-butylalcohol
第1次（0 h）The first time	4.0	5.0	1.0
第2次（1~ 4 h）The second time	3.0	5.0	2.0
第3次（1~ 4 h）The third time	1.5	5.0	3.5
第4次（1~ 4 h）The fourth time	0	5.0	5.0
第5次（1~ 4 h）The fifth time	0	2.5	7.5
第6次（0.5 ~ 2 h）The sixth time	0	0	10.0
第7次（0.5 ~ 2 h）The seventh time	0	0	10.0

Table 2 Dipping Procedure of wax for samples

程序 Procedure	各成分占比例The proportion of each component		石蜡温度/℃ Temperature of paraffin
程序 Procedure	叔丁醇Tert-butylalcohol	石蜡Paraffin	石蜡温度/℃ Temperature of paraffin
第1次The first time	2/3	1/3	48
第2次The second time	1/2	1/2	48
第3次The third time	1/3	2/3	48
第4次The fourth time	0	1/1	48
第5次The fifth time	0	1/1	54
第6次The sixth time	0	1/1	58

Fig. 1 Sequence information of restriction sites used in probe preparation The value in parentheses refers to the position of the base.

Table 3 Primers used in qPCR detection of Huanglongbing disease

引物 Primer	引物序列（5′-3′） Primer sequences	片段长度/bp Fragment length	GenBank 编号 GenBank Location accessory	参考文献 Reference
COX+	gtatgccacgtcgcattccaga	68	CX297817 72-93	Li et al.，2006
COX-	gccaaaactgctaagggcattc	68	CX297817 118-139	Li et al.，2006
RNRf	catgctccatgaagctaccc	80	CP010804.1 5434-5455	Zheng et al.，2016
RNRr	ggagcatttaaccccacgaa	80	CP010804.1 5493-5514	Zheng et al.，2016

Fig. 2 Alignment of five ribonucleotide reductase β subunit gene（nrdB）related sequences in Candidatus Liberibacter asiaticus strains psy62

Fig. 3 Sequencing results during probe preparation（a）and antisense strand gel electrophoresis of RNA（b） M：RNA marker;1：Anti-sense RNA probe of special fragment of ribonucleotide reductase（RNR）β subunit;2：Sense RNA probe.

Fig. 4 Expression characteristics of RNR gene in phloem of citrus petiole A：On petiole sections of‘Aisha'hybrid（Citrus reticulata‘Ehime 38'× C. reticulata Blanco）infected by Huanglongbing disease，hybridization with RNA antisense strand probe it was showed blue signal in sieve tube of phloem（150×）;B：In the control，no blue hybridization signal was generated in phloem with RNA sense probe hybridization（150×）;C：At a magnification of 500 times，the hybridization signal presented blue granules of varying sizes;D：Under an Olympus BX63 microscope，the blue hybridization signal was clearly visible in the sieve tube and companion cell（eyepiece 10×，objective 20×）. s：Sieve tube;c：Companion cell;ph：Phloem;xy：Xylem.

Fig. 5 In situ hybridization signals of RNA antisense strand probes in longitudinal branch peel section（A），petiole crosscut slice（B），vein longitudinal section（C），and root tip longitudinal section（D）of‘Aisha' A and D were observed by VHX-6000 ultra-depth of field three-dimensional microscopic system，B and C were observed by Olympus CX23 microscope. Ph：Phloem;xy：Xylem.

Fig. 6 In situ hybridization signals of RNA antisense strand probes in longitudinal section（A），longitudinal section（B），longitudinal section of petiole（C）and longitudinal section of stem tip（D）of‘Nanfeng Mandarin SS-28' G and H were observed by VHX-6000 ultra-depth of field three-dimensional microscopic system，E and F were observed by Olympus CX23 microscope. ph：Phloem;xy：Xylem;lp：Leaf primordium;m：Meristem.

Table 4 Relative expression levels of RNR genes in phloem bacilli in different organs of citrus

品种 Cultivar	样品 Sample	CT_RNR	CT_COX	∆∆CT	∆∆CT平均值 ∆∆CT mean	2^-∆∆CT	黄龙病 HLB
对照Control	阴性对照Negative control	28.583 ± 0.180	14.517 ± 0.157	0	0	1	-
对照Control	阳性对照Positive control	31.964 ± 0.944	23.549 ± 0.270	-5.421	-5.651	50.25	+
爱莎Aisha	枝皮Bark	29.362 ± 1.713	16.886 ± 0.435	-2.963	-1.59	3.01	+
	叶脉Veins	29.011 ± 2.202	17.054 ± 0.471	1.710	-2.108	4.31	+
	根Root	30.834 ± 1.418	15.240 ± 0.294	3.593	1.529	0.35	-
	茎尖Shoot tip	33.835 ± 1.728	18.306 ± 0.110	3.764	1.463	0.36	-
	叶柄Petiole	28.047 ± 3.002	14.748 ± 0.110	-4.645	-0.767	1.70	+
南丰蜜橘SS-28 Nanfeng Mandarin SS-28	枝皮Bark	28.845 ± 1.115	16.691 ± 0.218	-3.006	-1.912	3.76	+
	叶脉Veins	31.175 ± 1.616	17.277 ± 0.044	-0.028	-0.167	1.12	+
	根Root	34.692 ± 0.280	16.536 ± 0.438	3.304	4.091	0.06	-
	茎尖Shoot tip	33.298 ± 1.978	16.476 ± 0.072	4.108	2.757	0.15	-
	叶柄Petiole	34.349 ± 1.144	16.409 ± 0.688	4.703	3.875	0.07	-

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In Situ Hybridization Analysis of Distinctive Ribonucleotide Reductase β Subunit Gene from Candidatus Liberibacter Asiaticus of Citrus

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