| Non-heading Chinese cabbage belongs to the genus Brassica of Brassicaceae and plays an important role in China’s vegetable consumption market.The downy mildew caused by Hyaluronospora parasitica is one of the main diseases affecting the cruciferous crops.Under suitable environmental conditions,the disease can be re-infected repeatedly.The most frequently occurring period is early spring and late autumn.If the disease is not treated,the production will be reduced by 20%～60%.In order to enhance the disease resistance of non-heading Chinese cabbage,it is currently the most effective method to cultivate resistant varieties and master the disease resistance mechanism at the molecular biology level.In this study,the functional verification of BcPR-5L and BcCNL genes was performed,and the whole plant genome association analysis was used to identify the downy mildew resistance genes of non-heading Chinese cabbage.The results are as follows:1、Molecular cloning and characterization of a PR-5L like protein gene from non-heading Chinese cabbage(Brassica campestris ssp.chinensis Makino)In this study,a pathogenesis-related 5-like(PR-5L)protein gene,named BcPR-SL,was successfully cloned from non-heading Chinese cabbage.The cDNA sequence of BcPR-5L is 747 bp in length,encoding 248 amino acids with a protein molecular weight of 25.78 kDa and an isoelectric point of 4.42.Multiple sequence alignment indicated that BcPR-5L protein was highly homologous to other PR-5L proteins identified in 13 different species,with the highest homology to Brassica rapa.I analyzed the subcellular localization of BcPR-5L protein by using onion epidermal cells and found that it is localized in the membrane.Real time quantitative PCR analyses revealed that the expression of BcPR-5L gene was significantly upregulated after H.parasitica infection,and the expression level in the resistant varieties was higher than that in the susceptible varieties.2、Cloning and functional analysis of BcCNL gene in non-heading Chinese cabbageThe full-length cDNA of BcCNL was cloned using the cDNA of ’Suzhouqing’ as a template.The results showed that the BcCNL gene contained a 2790 bp open reading frame;the evolutionary relationship between BcCNL protein and CNL protein of Brassica oleracea var.oleracea is the most recent and contains RX-CC,NB-ARC,and LRR-3 domains.The subcellular localization of BcCNL protein was located in the cell membrane.At the same time,the expression of BcCNL gene in ’Suzhouqing’ was higher than that of’Aijiaohuang’ after infected with downy mildew.Silencing of BcCNL gene reduced the’Suzhouqing’ against downy mildew.3、A genome-wide association analysis of non-heading Chinese cabbage in resistance to downy mildewA genome-wide association analysis method was used to search for resistance to downy mildew in 102 non-heading Chinese cabbages.The results showed that the group can be divided into two subgroups,each of which contains a variety of non-heading cabbage varieties.Linkage disequilibrium analysis showed that when the r2 value was 0.18,the attenuation distance of the group was the shortest at 25 kb.When the threshold value was P=5,a candidate gene,Bra001654,was identified,which was located at 17729778 on the A03 chromosome.When the threshold value was P=3,five candidate genes could be identified,among which Bra036368 gene is located at 21127202 on the A02 chromosome.;Bra018253,located at 6960857 on the A05 chromosome;Bra030815,located at 57170 on the A08 chromosome;Bra036764,located at 7214909 on the A08 chromosome;Bra008888,located at 13100861 on the A10 chromosome.Through expression analysis in tissues,it was found that these six genes were expressed in roots and leaves,and were significantly expressed in leaves.It is speculated that the above six genes play an important role in the research of non-heading Chinese cabbage against downy mildew.These six genes were verified by real-time PCR,and it was found that in addition to the Bra030815 gene,the expression levels of Bra001654,Bra018253,Bra008888,Bra036764,and Bra036368 increased after inoculation with downy mildew. |
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