梭梭14-3-3蛋白HaFT-9在高温-干旱胁迫信号交叉调控中的功能研究

doi:10.11686/cyxb2024403

摘要/Abstract

摘要：

先前的研究表明，梭梭幼苗在生长过程中通过胁迫锻炼而获得高温、干旱等胁迫耐受性，并且14-3-3蛋白基因HaFT-9的表达受到高温和干旱胁迫的诱导，提示HaFT-9可能参与高温与干旱胁迫信号的交叉调控。为此，本研究探讨了HaFT-9在高温与干旱胁迫交叉调控作用中的功能。通过对梭梭幼苗进行高温胁迫锻炼后，再施加干旱胁迫，发现高温胁迫锻炼显著促进了HaFT-9在次级干旱胁迫下的表达。进一步的试验表明，经过高温胁迫锻炼与次级干旱胁迫处理，HaFT-9过表达的拟南芥株系表现出比野生型更高的存活率和更低的细胞死亡率。在次级干旱胁迫条件下，HaFT-9过表达株系中P5CS1、P5CS2、CAT2、CHLI1、HSP21、HsfA2及BI-1等抗逆相关基因的表达水平均显著升高。同时，过表达株系中的过氧化氢酶（CAT）活性、脯氨酸和叶绿素含量也显著增加，丙二醛（MDA）含量则显著降低，而且过表达株系的二氨基联苯胺（DAB）与硝基氮蓝四唑（NBT）染色较浅。综上所述，HaFT-9的过表达显著增强了拟南芥对次级干旱胁迫的适应性，表明HaFT-9在高温与干旱胁迫信号交叉响应中发挥了关键的调控作用。本研究为深入理解梭梭幼苗的耐逆分子机制提供了理论依据，并为保护其种质资源提供了参考。

关键词: 梭梭, 高温, 干旱, 交叉调控, HaFT-9

Abstract:

Previous research has demonstrated that Haloxylon ammodendron seedlings acquire resistance to stressors like high temperatures and drought through adaptive stress conditioning during their growth. Additionally， the expression of the 14-3-3 protein gene HaFT-9 is significantly upregulated in response to high temperature and drought stress， suggesting a potential role in mediating crosstalk between these stress pathways. This study further investigates the role of HaFT-9 in the cross-regulation of high temperature and drought stress signals. Pre-treatment of H. ammodendron seedlings with heat stress， followed by a subsequent drought stress challenge， significantly increased HaFT-9 expression during drought stress. Comparative experiments demonstrated that Arabidopsis thaliana lines overexpressing HaFT-9 showed higher survival rates and reduced cellular mortality under sequential heat and drought stress compared to wild-type controls. Notably， the lines overexpressing HaFT-9 exhibited enhanced expression of several key stress-responsive genes， including P5CS1， P5CS2， CAT2， CHLI1， HSP21， HsfA2 and BI-1， in response to subsequent drought stress.In addition， these lines showed significantly enhanced catalase （CAT） activity， increased proline and chlorophyll levels， while the content of malondialdehyde （MDA） was significantly reduced. Moreover， staining with diaminobenzidine （DAB） and nitroblue tetrazolium （NBT） in overexpressing lines was lighter. In conclusion， HaFT-9 overexpression markedly improves drought tolerance in A. thaliana， underscoring its critical regulatory role in modulating the interplay between high temperature and drought stress responses. This study provides valuable insights into the molecular mechanisms underlying stress tolerance in H. ammodendron seedlings and provides data relevant to the conservation of its germplasm resources.

Key words: Haloxylon ammodendron, high temperature, drought, cross regulation, HaFT-9

刘媛媛, 王旭, 魏琪, 车丽娟, 袁梦, 王波. 梭梭14-3-3蛋白HaFT-9在高温-干旱胁迫信号交叉调控中的功能研究[J]. 草业学报, 2025, 34(9): 134-146.

Yuan-yuan LIU, Xu WANG, Qi WEI, Li-juan CHE, Meng YUAN, Bo WANG. The role of HaFT-9， a 14-3-3 protein from Haloxylon ammodendron， in the cross regulation of high temperature and drought stress[J]. Acta Prataculturae Sinica, 2025, 34(9): 134-146.

图/表 7

表1 本研究所用引物

Table 1 Primers used in this study

基因 Gene	正向引物Forward primer （5′→3′）	反向引物 Reverse primer （5′→3′）
Ha18SrRNA	CTCTGCCCGTTGCTCTGATGAT	CCTTGGATGTGGTAGCCGTTTC
HaFT-9	TACTTTGGGTGAAGAGTCCTACA	TTTGATCCTGCATGTCTGATGT
P5CS1	GATACGGATATGGCAAAGCG	CCAAGTCCAAATCGGAAACC
P5CS2	GTTAAGCGTATCGTCGTCAAGGTT	CCTAAACGTCCAAGAGCCAATCT
CAT2	ACATTCGTTGACCCTTTTCAC	GGCCACACACGATAACAAC
CDS1	CTGCATCTCTACTGGACCTC	CCACATCCAACTCTCGAGC
CHLI1	TGTTGATGGGTTGAGAGGAG	AACGGTTGCAACATCATCTG
DREB2a	CAACAGCAGGATTCGCTATCTG	ACATCGTCGCCATTTAGGTCA
HSP21	TGGACGTCTCTCCTTTCGGATTGT	TGCACGAATCTCTGACACTCCACT
HsfA2	GAGATTTTCCGGCGTGATTT	ACATCGGACCTGAAAGAGAG
BI-1	ACATTCGTTGACCCTTTTCAC	GGCCACACACGATAACAAC
At18SrRNA	AAACGGCTACCACATCCAAG	CCTCCAATGGATCCTCGTTA

图1 HaFT-9基因在高温胁迫锻炼-次级干旱胁迫处理下的表达A：梭梭幼苗的高温胁迫锻炼-次级干旱胁迫处理，分为CK、H、D、HD组，每个红框表示取样点；B：HaFT-9在高温胁迫锻炼-次级干旱胁迫处理下的表达量；不同小写字母表示在P<0.05水平上差异显著。A： The heat stress priming-the secondary drought stress treatment for H. ammodendron seedlings， divided into CK， H， D and HD group， and each red box indicates a sampling point； B： Expression level of HaFT-9 under the heat stress priming-the secondary drought stress treatment； Different lowercase letters indicate significant differences at P<0.05level. 下同The same below.

Fig.1 The expression of the HaFT-9 gene under the heat stress priming-the secondary drought stress treatment

图2 高温胁迫锻炼-次级干旱胁迫处理对拟南芥生长的影响A：拟南芥幼苗的高温胁迫锻炼-次级干旱胁迫处理，分为CK、H、D、HD组，每个红框表示取样点；B：拟南芥成株的高温胁迫锻炼-次级干旱胁迫处理，分为CK、H、D、HD组，每个红框表示取样点；C：拟南芥幼苗的生长表型；D：拟南芥成株的生长表型；WT为野生型拟南芥，9-4和9-37为HaFT-9过表达株系。A： The heat stress priming-the secondary drought stress treatment for Arabidopsis seedlings， divided into group CK， H， D and HD， and each red box indicates a sampling point； B： The heat stress priming-the secondary drought stress treatment for mature Arabidopsis， divided into CK， H， D and HD group， and each red box indicates a sampling point； C： Growth phenotype of Arabidopsis seedlings； D： Growth phenotype of mature Arabidopsis； WT refers to the wild-type Arabidopsis， while 9-4 and 9-37 are the HaFT-9 overexpression lines. 下同The same below.

Fig.2 The effects of the heat stress priming-the secondary drought stress treatment on the growth of Arabidopsis

图3 高温胁迫锻炼-次级干旱胁迫处理对拟南芥存活率及细胞死亡率的影响A：拟南芥幼苗台盼蓝染色（叶片，标尺=50 μm）；B：拟南芥幼苗台盼蓝染色后叶片的细胞死亡率；C：拟南芥幼苗的存活率；D：拟南芥幼苗DAPI染色（根尖，标尺=50 μm）；E：拟南芥幼苗DAPI染色后的根尖细胞死亡率；F：拟南芥成株的存活率。A： Trypan blue staining in Arabidopsis seedlings （leaf， bar=50 μm）； B： Cell death rate of leaves in Arabidopsis seedlings after Trypan blue staining； C： Survival rate of Arabidopsis seedlings； D： DAPI staining in Arabidopsis seedlings（root tip， bar=50 μm）； E： Cell death rate in the root tips of Arabidopsis seedlings after DAPI staining； F： Survival rate of mature Arabidopsis.

Fig.3 The effects of the heat stress priming-the secondary drought stress treatment on the survival rate and cell death rate in Arabidopsis

图4 拟南芥在高温胁迫锻炼-次级干旱胁迫处理下的相关基因表达A：拟南芥幼苗在高温胁迫锻炼-次级干旱胁迫处理下的相关基因表达；B：拟南芥成株在高温胁迫锻炼-次级干旱胁迫处理下的相关基因表达。A： The expression of related genes in Arabidopsis seedlings under the heat stress priming-the secondary drought stress treatment； B： The expression of related genes in mature Arabidopsis under the heat stress priming-the secondary drought stress treatment.

Fig.4 The expression of relevant genes under the heat stress priming-the secondary drought stress treatment in Arabidopsis

图5 高温胁迫锻炼-次级干旱胁迫处理下拟南芥的二氨基联苯胺（DAB）与硝基氮蓝四唑（NBT）染色A、B：分别为拟南芥幼苗在高温胁迫锻炼-次级干旱胁迫处理下的二氨基联苯胺（DAB）和硝基氮蓝四唑（NBT）染色（标尺=100 μm）；C、D：分别为拟南芥成株在高温胁迫锻炼-次级干旱胁迫处理下的二氨基联苯胺（DAB）和硝基氮蓝四唑（NBT）染色（标尺=2.5 mm）。A， B： Diaminobenzidine （DAB） and nitroblue tetrazolium （NBT） staining of Arabidopsis seedlings under the heat stress priming-the secondary drought stress treatment， respectively （Bar=100 μm）； C， D： Diaminobenzidine （DAB） and nitroblue tetrazolium （NBT） staining of mature Arabidopsis under the heat stress priming-the secondary drought stress treatment， respectively （Bar=2.5 mm）.

Fig.5 Diaminobenzidine （DAB） and nitroblue tetrazolium （NBT） staining of Arabidopsis under the heat stress priming-the secondary drought stress treatment

图6 高温胁迫锻炼-次级干旱胁迫处理下拟南芥的抗氧化酶活性、脯氨酸和叶绿素含量A：高温胁迫锻炼-次级干旱胁迫处理下拟南芥幼苗的抗氧化酶活性、脯氨酸和叶绿素含量；B：高温胁迫锻炼-次级干旱胁迫处理下拟南芥成株的抗氧化酶活性、脯氨酸和叶绿素含量。A： Antioxidant enzyme activity， proline content， and chlorophyll content under the heat stress priming-the secondary drought stress treatment in Arabidopsis seedlings； B： Antioxidant enzyme activity， proline content， and chlorophyll content under the heat stress priming-the secondary drought stress treatment in mature Arabidopsis.

Fig.6 Antioxidant enzyme activity， proline content， and chlorophyll content under the heat stress priming-the secondary drought stress treatment in Arabidopsis

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