Loss-of-function mutations of genes encoding BnaBIN2 kinases enhance cold tolerance of <italic style="font-style: italic">Brassica napus</italic> seedling

ZHANG Bin-yuan; ZHENG Yue-ping; LIU Hong-bo; ZHENG Zhi-fu

doi:10.19802/j.issn.1007-9084.2026147

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Loss-of-function mutations of genes encoding BnaBIN2 kinases enhance cold tolerance of Brassica napus seedling

更新时间：2026-10-31

- Loss-of-function mutations of genes encoding BnaBIN2 kinases enhance cold tolerance of Brassica napus seedling
- Chinese Journal of Oil Crop Sciences Vol. 48, Issue 4, Pages: 91-103(2026)
- 作者机构：
  
  1.浙江农林大学现代农学院，浙江杭州，311300
  2.油料作物种质创新与利用研究所，浙江杭州，311300
- 作者简介：
- 基金信息：
- DOI：10.19802/j.issn.1007-9084.2026147
  CLC： S565.4
- Received：07 May 2026，
  
  Published：25 August 2026
- 稿件说明：
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张滨元,郑月萍,刘宏波等.激酶编码基因BnaBIN2的功能缺失突变增强油菜幼苗耐冷性[J].中国油料作物学报,2026,48(04):91-103.

ZHANG Bin-yuan,ZHENG Yue-ping,LIU Hong-bo,et al.Loss-of-function mutations of genes encoding BnaBIN2 kinases enhance cold tolerance of Brassica napus seedling[J].Chinese Journal of Oil Crop Sciences,2026,48(04):91-103.
张滨元,郑月萍,刘宏波等.激酶编码基因BnaBIN2的功能缺失突变增强油菜幼苗耐冷性[J].中国油料作物学报,2026,48(04):91-103. DOI： 10.19802/j.issn.1007-9084.2026147.

ZHANG Bin-yuan,ZHENG Yue-ping,LIU Hong-bo,et al.Loss-of-function mutations of genes encoding BnaBIN2 kinases enhance cold tolerance of Brassica napus seedling[J].Chinese Journal of Oil Crop Sciences,2026,48(04):91-103. DOI： 10.19802/j.issn.1007-9084.2026147.

摘要

低温胁迫会导致油菜产量的损失，油菜素内酯在低温应答过程中发挥重要作用。本研究的目的是剖析油菜素甾醇（brassinosteroids，BRs）信号通路中的BnaBIN2激酶对油菜低温耐受性的调控作用。全基因组分析显示，甘蓝型油菜存在6个编码BnaBIN2的基因，这些激酶均含有STKc_GSK3结构域；尽管这些激酶的氨基酸序列相似性达96.3%，但相应基因的启动子区域的顺式作用元件存在差异，暗示这些同源基因可能存在功能冗余与功能分化。RT-qPCR分析显示，所有

BnaBIN2

基因在各组织中呈现广泛表达特征，且均受低温诱导，但它们对低温响应的强度存在一定差异，

BnaA03.BIN2、BnaA05.BIN2、BnaC01.BIN2

表达量上调约8倍，

BnaA01.BIN2、BnaC06.BIN2

约6倍，而

BnaC03.BIN2

不足3倍。为进一步揭示

BnaBIN2

的功能，本研究运用基因编辑

技术创制了

BnaBIN2

基因的单、五和六重功能丧失型突变体，发现这些突变体的耐冷性显著高于野生型。生理生化分析显示，突变体的二氨基联苯胺染色较浅，活性氧积累量减少；相应地，突变体的超氧化物歧化酶和过氧化物酶活性则显著高于野生型。同时，突变体的脯氨酸和可溶性糖含量增加，而相对电导率和丙二醛含量则显著低于野生型。上述结果表明，

BnaBIN2

基因突变增强了细胞膜稳定性和活性氧清除能力，从而提高了油菜的耐冷性。这些突变体有望用于油菜耐冷性状的遗传改良。

Abstract

Low temperature stress can lead to the loss of rapeseed yield

and brassinolide plays an important role in the process of low temperature response. The purpose of this study was to dissect the regulatory roles of BnaBIN2 kinases of the brassinosteroid (BR) signaling pathway in low temperature tolerance of rapeseed. Genome-wide analysis showed that there were six genes encoding BnaBIN2 kinases in

napus

all of which contained STKc_GSK3 domain. Although these kinases share 96.3% of sequence similarity at the amino acid level

the corresponding genes vary in the

cis

-acting elements in the promoter regions

implying the possible existence of functional redundancy and functional divergence of homologous

BnaBIN2

genes. RT-qPCR analysis showed that although all

BnaBIN2

genes were ubiquitously expressed in all tissues tested and induced by low temperature

the extent to which they respond to low temperature appeared to be different. The expression levels of

BnaA03.BIN2

BnaA05.BIN2

and

BnaC01.BIN2

showed an approximately 8-fold increase after cold treatment

whereas only about a 6-fold increase for

BnaA01.BIN2

and

BnaC06.BIN2

and a less than 3-fold increase for

BnaC03.BIN2

were registered. To further elucidate the function of

BnaBIN2s

gene editing technology was employed to generate single

quintuple

sextuple loss-of-function mutants of

BnaBIN2

genes. It was found that the cold tolerance of these mutants was significantly higher than that of wild type. Physiological and bi

ochemical analysis showed that the mutants had weaker DAB staining and reduced accumulation of reactive oxygen species relative to the wild type (WT). Accordingly

the activities of superoxide dismutase and peroxidase in the mutants were significantly higher than those in WT plants. Meanwhile

the contents of proline and soluble sugar increased in the mutants

whereas the opposite trend was seen for the relative electrical conductivity and malondialdehyde content. Collectively

these results indicate that the mutations of

BnaBIN2

enhance the stability of cell membrane and the capacity of scavenging reactive oxygen species

thereby improving rapeseed cold tolerance. These mutants are expected to be useful in genetic improvement of cold tolerance trait of rapeseed.

关键词

Keywords

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Loss-of-function mutations of genes encoding BnaBIN2 kinases enhance cold tolerance of Brassica napus seedling

DOI：10.19802/j.issn.1007-9084.2026147

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