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Journal of Integrative Agriculture 2014, 13(11): 2407-2415 November 2014 RESEARCH ARTICLE © 2014, CAAS. All rights reserved. Published by Elsevier Ltd. doi: 10.1016/S2095-3119(13)60696-6 Over-Expression of BnMAPK1 in Brassica napus Enhances Tolerance to Drought Stress WENG Chang-mei 1, 2, 3 , LU Jun-xing 1, 2, 3 , WAN Hua-fang 1, 2, 3 , WANG Shu-wen 1, 2, 3 , WANG Zhen 1, 2, 3 , LU Kun 1, 2, 3 and LIANG Ying 1, 2, 3 1 College of Agronomy and Biotechnology, Southwest University, Chongqing 400715, P.R.China 2 Chongqing Rapeseed Engineering & Technology Research Center, Chongqing 400715, P.R.China 3 Engineering Research Center of South Upland Agriculture, Ministry of Education, Chongqing 400715, P.R.China Abstract Mitogen-activated protein kinases (MAPKs) are a family of Ser/Thr protein kinases widely conserved in all eukaryotes and involved in responses to biotic and abiotic stresses. In this study, two over-expressing BnMAPK1 oilseed rape lines, ov3 and ov11, were used to study the drought-resistant mechanism of BnMAPK1 under natural drought and simulation drought through spraying 10% PEG 8000 in seedlings. Zhongyou 821 (WT) was used as control. Compared with wild type, transgenic seedlings had higher leaf water content, higher root activity, slightly higher peroxidase (POD) and superoxide dismutase (SOD) activity, higher proline content and lower malondialdehyde (MDA) content. The expression of drought-resistant related genes, including P5CSB, PLC, LEA4 and SCE1, have been up-regulated in some degree and the expressed time of transgenic lines were earlier than that of wild type. These results suggested that over-expression of BnMAPK1 can enhance the resistance to drought in oilseed rape (Brassica napus). Key words: MAPK, Brassica napus, transgenic, drought-stress, qRT-PCR INTRODUCTION Oilseed rape (Brassica napus) is one of the main oil crops in China, and supplies more than half of edible oil (Yang and Xu 2010). However, oilseed rape often suffers from many stresses, such as cold, salinity and drought (Boyer 1982), which cause great yield loss ev- ery year (Dai et al. 2006). Among these stresses, water deficiency is a major abiotic factor (Manavalan et al. 2009). Drought stress has been known to induce many morphological, biochemical and molecular alterations that negatively affect plant growth and productivity (Wang et al. 2001). It is necessary to study the drought tolerance mechanism of plant from morphological to molecular level for improving crop yields. Mitogen-activated protein kinases (MAPKs) are wide- ly found in plant, and they performed via cascades which consist of three sequentially activated kinases, MAP kinase (MAPK), MAPK kinase (MAPKK), and MAPKK kinase (MAPKKK) (Zhang and Liu 2002). MAPKs are phosphorylated and activated by MAPK-kinases (MAPKKs), which are phosphorylated and activated by MAPKK-kinases (MAPKKKs). The MAPKKKs are activated by interaction with the family of small GTPases and/or other protein kinases, connecting the MAPK module to cell surface receptors or external stimuli. The activation and phosphorylation of MAPK can lead to changes in its subcellular localization and Received 28 October, 2013 Accepted 16 December, 2013 WENG Chang-mei, E-mail: [email protected]; Correspondence LIANG Ying, E-mail: [email protected]

Over-Expression of BnMAPK1 in Brassica napus Enhances Tolerance to Drought Stress

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Page 1: Over-Expression of BnMAPK1 in Brassica napus Enhances Tolerance to Drought Stress

Journal of Integrative Agriculture2014, 13(11): 2407-2415 November 2014RESEARCH ARTICLE

© 2014, CAAS. All rights reserved. Published by Elsevier Ltd.doi: 10.1016/S2095-3119(13)60696-6

Over-Expression of BnMAPK1 in Brassica napus Enhances Tolerance to Drought Stress

WENG Chang-mei1, 2, 3, LU Jun-xing1, 2, 3, WAN Hua-fang1, 2, 3, WANG Shu-wen1, 2, 3, WANG Zhen1, 2, 3, LU Kun1, 2, 3 and LIANG Ying1, 2, 3

1 College of Agronomy and Biotechnology, Southwest University, Chongqing 400715, P.R.China2 Chongqing Rapeseed Engineering & Technology Research Center, Chongqing 400715, P.R.China3 Engineering Research Center of South Upland Agriculture, Ministry of Education, Chongqing 400715, P.R.China

Abstract

Mitogen-activated protein kinases (MAPKs) are a family of Ser/Thr protein kinases widely conserved in all eukaryotes and involved in responses to biotic and abiotic stresses. In this study, two over-expressing BnMAPK1 oilseed rape lines, ov3 and ov11, were used to study the drought-resistant mechanism of BnMAPK1 under natural drought and simulation drought through spraying 10% PEG 8000 in seedlings. Zhongyou 821 (WT) was used as control. Compared with wild type, transgenic seedlings had higher leaf water content, higher root activity, slightly higher peroxidase (POD) and superoxide dismutase (SOD) activity, higher proline content and lower malondialdehyde (MDA) content. The expression of drought-resistant related genes, including P5CSB, PLC, LEA4 and SCE1, have been up-regulated in some degree and the expressed time of transgenic lines were earlier than that of wild type. These results suggested that over-expression of BnMAPK1 can enhance the resistance to drought in oilseed rape (Brassica napus).

Key words: MAPK, Brassica napus, transgenic, drought-stress, qRT-PCR

INTRODUCTION

Oilseed rape (Brassica napus) is one of the main oil crops in China, and supplies more than half of edible oil (Yang and Xu 2010). However, oilseed rape often suffers from many stresses, such as cold, salinity and drought (Boyer 1982), which cause great yield loss ev-ery year (Dai et al. 2006). Among these stresses, water deficiency is a major abiotic factor (Manavalan et al. 2009). Drought stress has been known to induce many morphological, biochemical and molecular alterations that negatively affect plant growth and productivity (Wang et al. 2001). It is necessary to study the drought

tolerance mechanism of plant from morphological to molecular level for improving crop yields.

Mitogen-activated protein kinases (MAPKs) are wide-ly found in plant, and they performed via cascades which consist of three sequentially activated kinases, MAP kinase (MAPK), MAPK kinase (MAPKK), and MAPKK kinase (MAPKKK) (Zhang and Liu 2002). MAPKs are phosphorylated and activated by MAPK-kinases (MAPKKs), which are phosphorylated and activated by MAPKK-kinases (MAPKKKs). The MAPKKKs are activated by interaction with the family of small GTPases and/or other protein kinases, connecting the MAPK module to cell surface receptors or external stimuli. The activation and phosphorylation of MAPK can lead to changes in its subcellular localization and

Received 28 October, 2013 Accepted 16 December, 2013WENG Chang-mei, E-mail: [email protected]; Correspondence LIANG Ying, E-mail: [email protected]

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its interaction with transcriptional factors, and thereby reprogram gene expression (Fiil et al. 2009). At least 20 MAPKs have been identified and studied in Arabi-dopsis (Ichimura et al. 2002) and a variety of MAPK genes have also been identified in rice (Reyna and Yang 2006), poplar (Nicole et al. 2006), and oilseed rape (Yu 2004; Yin et al. 2008; Zhu et al. 2013). More and more evidences have shown that the MAPKs are involved in response to various biotic and abiotic stresses such as salinity, drought, cold and pathogens (Jonak et al. 1996; He et al. 1999; Mikolajczyk et al. 2000).

According to the conserved amino acid sequence of TXY motif in MAPKs, MAPKs can be classified into two groups in plants, namely TEY, including A, B and C subgroups, and TDY, including D subgroup (Ichimura et al. 2002; Hamel et al. 2006). Previous studies have mainly focused on group A and B MAPKs (Ichimura et al. 2000; Yin et al. 2008; Beckers et al. 2009), whereas the information on group C and D MAPKs are relatively lim-ited. The increasing evidences have revealed that group C MAPKs, such as AtMPK1, AtMPK2, PsMPK2, GhMPK2, ZmMPK7 et al. are involved in various signaling processes and have unique biological functions (Ortiz-Masia et al. 2007, 2008; Zong et al. 2009; Liang et al. 2011). The objective of this study is to verify whether BnMAPK1 are involved in response to drought stress in B. napus via assaying T2 of transgenic plants. The expression profile of several genes including P5CSB, PLC, LEA4, SCE1 which were likely to involve in signal pathway or regulation of drought stress were detected (Hirayama et al. 1995; Kishor et al. 1995; Park et al. 2005; Xiao et al. 2007; Karan and Subudh 2012), and biochemical factor antioxidase activity (POD, peroxidase; SOD, superoxide dismutase; CAT, catalase), root activity, content of malondialdehyde (MDA) and proline were measured both in BnMAPK1 over-expressing transgenic lines and wild type B. napus. This study will provide reference for understanding the function of plants MAPK1, and offer theoretical basis for oilseed rape resistance breeding.

RESULTS

The changes of soil water content and leaf water content during drought stress

At the six-leaf stage, all the experimental materials

were exposed to drought stress. At the beginning, the soil water content was approximately at 50%, which decreased quickly after the limited water supply. After the 8-d duration of drought, soil water content (SWC) decreased to 15%, indicating it was droughty (Chen et al. 2009). Thereafter, SWC had no distinct change until the experiment finished, namely 12 d after the treatment. During the treatment, the SWC with WT had no significant difference from that with transgenic plants, ov3 and ov11 (Fig. 1-A). At the early stage of treatment, the change of water content in the leaves was not obvious in both the WT and the transgenic plants. It varied from 91 to 88% and 92 to 90% in WT plants and the transgenic lines, respectively. With the extending of drought, especially at the later stage (8 to 12 d), the leaf water in WT declined drastically to 83%, which was significantly lower than that of the transgenic plants with 90-88% (Fig. 1-B). It showed that the over-expression of BnMAPK1 alleviated the dehydration in the leaf of

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Fig. 1 Changes of water content in the soil (A) and the leaves (B) of WT and transgenic (ov3 and ov11) plants during natural drought stress.WT is wild type; ov3 and ov11 were two BnMAPK1 over-expression transgenic lines. Values are means±SE (n=3). B, bars carrying one asterisk (*) are significantly different level at P=0.05 and bars carrying two asterisks (**) are significantly different level at P=0.01 compared with WT. The same as below.

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B. npaus when exposed to drought.

Biochemical and physiological characteristics of B. napus under drought stress

Over-expression of BnMAPK1 on root activity of B. napus seedlings exposed to drought stress As it was shown in Fig. 2, the change pattern of root activity in BnMAPK1 over-expressing transgenic and WT B. napus seedlings under drought stress was similar. The activity immediately increased in the initial period of treatment followed by a decrease with the extension of treatment. Interestingly, the root activity of the transgenic plants was significantly higher than that of the control under drought stress or not. Additionally, with the extension of the treatment, the difference of the root activity between the transgenic plants and the wild types became bigger and bigger. All the results indicated that BnMAPK1 gene was likely to promote the root activity, especially under drought stress, relieving the injury from drought stress.

Fig. 3 displayed the growth and phenotype of Bn-MAPK1 over-expressing transgenic and WT B. napus seedlings after 12 d drought stress treatment. Fig. 3-A showed the seedlings grown in pots, and Fig. 3-B showed the whole plant of B. napus seedlings pulled from the pots. From Fig. 3-A, slighter wilting was found in transgenic plants ov3 and ov11, and grew better than WT. Fig. 3-B showed that the root system of transgenic plants was better developed and had more fibril than the wild types.Change of proline and MDA content under drought stress The background content of proline in WT and transgenic plants was low and with little difference. After the exposure to the drought stress, it increased with different extent in WT and transgenic lines, espe-cially at the later stage. The content of proline in ov3 and ov11 were 28-36 and 23-42 times higher than the background, respectively. However, the increase level in WT was lower compared with the transgenic ones, approximately 20 times. The maximum of proline in the transgenic plants was 3-4 times higher than that in the WT (Fig. 4-A). MDA was induced in all experimental materials, but the increase level was different in WT and transgenic plants. In the process, MDA content in transgenic plants was much lower than that in WT. And the difference was significant at 6 and 12 d under

drought stress (Fig. 4-B). All the results indicated that over-expression of BnMAPK1 effectively stimulated the accumulation of proline and alleviated oxidative damage of cell membrane in B. napus suffering from drought stress, resulting in the enhancement of resistance to drought.Activity of reactive oxygen species scavenging en-zymes under drought stress The activity of the anti-oxidant-enzymes, including POD, SOD and CAT, was strengthened at the beginning and followed with some fallback at the late stage during the drought treatment (Fig. 5-A, B and C). After 6-d treatment, the activity of all the enzymes reached to the maximum. In the exper-iment, the activity of POD and SOD in transgenic plants

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Fig. 2 Difference of root activity between BnMAPK1 over-expressing transgenic and WT B. napus plants exposed to natural drought stress.

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Fig. 3 Growth and phenotype of BnMAPK1 over-expressing transgenic and WT B. napus plants at 12 d after natural drought stress treatment. A showed the plants in the pots after 12 d drought, and B showed the whole plant after 12 d drought.

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was higher than that in WT, without distinct difference. However, the activity of CAT was similar in all the plants. All the results suggested that the over-expres-sion of BnMAPK1 had no significant influence on the antioxidant enzymes in B. napus.

qRT-PCR analysis of the genes related to drought response in B. napus under drought stress simulated with PEG8000

Expression of BnMAPK1 in B. napus under drought stress simulated with PEG8000 The expression of BnMAPK1 was distinctly different among the plants including transgenic and WT (Fig. 6). Under the stress, the expression in transgenic lines was significantly higher than that of WT, in which the expression was consistently low. Interestingly, a little difference occurred in the ex-pression profile between ov3 and ov11. The maximum expression level appeared at 9 h after the treatment in ov3, while it appeared at 1 h in ov11. It was 19 times and 44 times higher in ov3 and ov11 than that of WT, respectively. The expression profile of the BnMAPK1 gene showed that it responded to the simulated stress with high level of expression in transgenic lines.

Expression analysis of drought-resistant related genes respond to drought stress In all materials, the P5CSB gene and PLC gene were up-regulated under the drought stress. The maximum transcript accumulation of the two genes simultaneously occurred at 1 h after the treatment in the two transgenic lines. And the expression of P5CSB was 2.1 and 1.6 times higher than that of the background in ov3 and ov11, respectively. Similarly, the expression of PLC increased 2.4 and 2 times, respectively. In the following treatment process, the expression gradually decreased. But at 24-h exposure to stress, the expression increased and was higher than that of WT. The maximum transcript accumulation of the genes mentioned above in WT appeared at 3 h after the treatment, later than that in the transgenic lines. The expression of P5CSB and PLC in WT at 3 h after exposure to drought was 2.7 and 1.9 times higher than that of the background. However, the expression decreased gradually (Fig. 7-A and B).

Fig. 4 Changes of proline (A) and MDA content (B) in the leaves of WT and transgenic (ov3 and ov11) plants during natural drought stress.

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Fig. 5 Changes of antioxidant enzyme activities in the leaves of WT and transgenic (ov3 and ov11) plants during natural drought stress. A, peroxidase (POD) activites. B, superoxide dismutase (SOD) activites. C, catalase (CAT) activites.

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The expression of the other genes, including LEA4 and SCE1, had a similar profile in transgenic lines and WT. Compared with the background, the expression of the two genes was up-regulated under the simulated drought stress. The highest expression of LEA4 and SCE1 ap-peared at 3 and 1 h after the treatment in the transgenic lines, respectively (Fig. 7-C and D). However, it de-creased at 9 h after the stress, higher than that of WT, in which only little change was found in the expression of the two genes.

To summary, BnMAPK1 timely responded to the drought stress and was up-regulated. P5CSB, PLC, LEA4 and SCE1 were induced by the drought stress. The response to the stress was faster in the transgenic lines than that in WT. All the data indicated that BnMAPK1 was involved in the adaptation to the drought stress and induced the genes related to drought resistance, which might improve the resistance to drought stress in B. npaus.

DISCUSSION

An increasing amounts of evidence showed that the MAPK-mediated cellular signaling is crucial to plant growth and development, as well as biotic and abiotic stresses responses (Zhu and Liang 2012). Various stresses threatening to the production of oilseed rape, which stimulated the scientists to improve the resis-tance via transgenic technology employing MAPK as the target genes. In the present study, using transgenic lines over-expressing BnMAPK1 as main experimental materials, several physiological index were measured and the expression of related genes involved in resis-

tance to drought were examined under natural drought and artificial stress simulated with PEG8000, in order to discover the role of the BnMAPK1 in enhancing drought tolerance of oilseed rape (B. napus). The results indicated that BnMAPK1 over-expressing B. napus had higher leaf water content and could maintain the met-abolic capacity and alleviate the injury from drought stress.

Fig. 6 The qRT-PCR analysis of BnMAPK1 in transgenic and wild type seedlings during PEG-simulated drought.

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Fig. 7 The qRT-PCR analysis of related genes in transgenic and wild type seedlings during PEG-simulated drought. A, qRT-PCR analysis of P5CSB. B, qRT-PCR analysis of PLC. C, qRT-PCR analysis of LEA4. D, qRT-PCR analysis of SCE1.

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Root is the main organ of plant absorbing mineral and water. The root activity influence the ability of absorp-tion of nutrients and water. When plants are exposed to drought stress, the roots received the stress signal firstly, and then lead to other physiological response such as photosynthesis and cellular metabolism, therefore, the roots of plants were related to drought tolerance (Hu et al. 2013). Gao et al. (2005) proved that the root activity of oilseed rape was related to drought tolerance and can be adopted as index of drought resistance identification. The study on soybean and maize showed that the root activity declined under the drought condition and the plants with strong drought resistance had higher root activity than plants with weak drought resistance (Liu et al. 2002; Dong et al. 2011). The results in this study showed that the root activity of BnMAPK1 over-ex-pressing transgenic plants was much higher than that of WT, especially under the drought stress treatment. This suggested that the BnMAPK1 could promote absorption of mineral and water in the roots of B. napus seedlings, and improve drought resistance to some extent.

Proline accumulation is one of the most widespread metabolic responses of plants to stress (Ghars et al. 2008; Cao et al. 2011). MDA is considered as a symbol of stress-induced damage at the cellular level (Jain et al. 2001; Türkan et al. 2005). The results of this study showed an increase level in the proline and decrease level in the MDA in leaves of the BnMAPK1 over-ex-pressing B. napus seedlings, compared with the wild type during experimental period under natural drought stress, which is similar to the former studies (Jiang et al. 1997; Xu et al. 2000; Türkan et al. 2005; Vendruscolo et al. 2007; Li et al. 2011). The results indicated that over-expression of BnMAPK1 was likely to participate in regulating osmotic substance biosynthesis and membrane lipid peroxidation.

As other environmental stresses including salinity, low and high temperatures, drought stress also leads to oxidative injury. The plants can biosynthesize an-tioxidants such as SOD, POD and CAT to scavenge the radicals and alleviated the damage from the stress (Chung et al. 2006; Misra and Gupta 2006; Feng et al. 2008). In this study, the activity of SOD, POD and CAT in both BnMAPK1 over-expressing transgenic lines and WT was all initially increased to the highest value at 6 d

during natural drought stress and followed by a decrease with the increasing stress duration. However, there was no significant difference between transgenic and WT seedlings, which inferred that maybe the BnMAPK1 responded to the regulation of radical scavenging indi-rectly, and alleviated cellular oxidation by modulating other pathway. The underlying mechanism is worth of studying further.

The expression of ∆1-pyrroline-5-carboxylate syn-thetase gene (P5CS), coding the key enzyme in the proline synthesis, can contribute to the accumulation of proline, which is important in osmotic adjustment for plants under stress. Kishor et al. (1995) and Hong et al. (2000) reported that strong tolerance was induced in transgenic tobacco seedlings with P5CS over-expres-sion against stresses such as high salinity and drought, water-stress. Phospholipase C is a key enzyme in plant adversity signal transduction. It has proved that PLC could be significantly induced by dehydration, salinity, and temperature. PLC played a key role in the develop-ment of thermotolerance in pea leaves (Hirayama et al. 1995; Liu et al. 2006). Late-embryogenesis-abundant proteins (LEA) are proteins in plants generating with seeds maturation, which protect other proteins from aggregation due to desiccation or osmotic stresses. It can be used as shelter from dehydration, adjusting other genes expression interacted with nucleic acid as regula-tion protein (Li et al. 2010). Over-expression of LEA in rice and Chinese cabbage significantly enhanced drought and salt resistance (Park et al. 2005; Xiao et al. 2007). SUMO (small ubiquitin related modifier) conjugation enzyme (SCE) is a kind of ubiquitin protein regulating protein at translation level, which can be induced by high temperature, high salinity and drought stress. Nigam et al. (2008) reported two SCE genes were induced by high temperature. Karan and Subudh (2012) discovered that over-expressing SaSce9 separating from Spartina alterniflora in Arabidopsis enhance the tolerance against salinity and drought stress.

Our results revealed that BnMAPK1 could respond to drought stress. Meanwhile, the expression of drought-re-sistant related genes, P5CSB, PLC, LEA4 and SCE1, has been up-regulated in some degree. And the expression maximum appeared earlier than that of wild type, sug-gesting that BnMAPK1 could enhance drought resistance in B. napus to some extent.

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CONCLUSION

BnMAPK1 could promptly respond to drought stress and induce the expression of related genes, P5CSB, PLC, SCE1 and LEA4, involve in synthesis of proline and leaf water metabolism, promote the growth of plant roots and increase the root activity, regulating cell membrane lipid peroxidation. BnMAPK1 was speculated to enhance the drought tolerance in B. napus to a certain degree.

MATERIALS AND METHODS

Plant materials

Two over-expressing BnMAPK1 transgenic lines (patent has passed the preliminary examination), designated as ov3 and ov11, and their adopter parental line, Zhongyou 821 (wild type) were chosen for studying drought tolerance. The plants were grown in phytotron under 16 h-light/8 h-dark regime with 75% relative humidity at 21°C.

More information about BnMAPK1 and materials were offered in the patent which has already passed the preliminary examination (201310389076.3) and Lu et al. (2013).

Assay of biochemical and physiological indexes

The plants were stopped to watering as natural drought at 6-leaf age. The leaves were sampled for measuring six physiological-biochemical traits involved in drought tolerance after 0, 2, 4,

6, 8, and 12 d of water-limit treatment, respectively. The fresh leaves (FW, 0.5-0.6 g) were exposed to 105°C for 10 min to inactivate the enzymes, and then dried to constant weight (DW) at 80°C. Leaf water content (LWC) was calculated according to the formula, LWC (%)=((FW-DW)/FW)×100, root activity was measured with triphenyltetrazolium chloride (TTC) method according to Zhang and Qu (2003),the content of MDA and proline were determined with the method of thiobarbituric acid (TBA) and acidic-ninhydrin, respectively (Li et al. 2000). The activity of SOD, POD and CAT was determined using Nanjing Jiancheng Assay Kits (Nanjing Jiancheng Company, Nanjing, China). Soil water content was detected by soil moisture measuring instrument (Zhejiang Top Apparatus Limited Company, Zhejiang, China).

All the data were analyzed using SPSS 18.0 and EXCLE 2003, and figures were drawn using EXCEL 2003.

qRT-PCR analysis

In order to detect the effect of the over-expression of BnMAPK1 on the resistance to drought stress, the expression profile of several genes, P5CSB, PLC, SCE1 and LEA4, involved in drought stress were detected by qRT-PCR analysis, RNA were isolated from the leaves at 4 leaf-age plants at 0, 1, 3, 9, 24 h after being sprayed with 10% PEG 8000, respectively. Total RNA was prepared and purified using the RNAprep Pure Plant Kit (Tiangen Biotech, Beijing, China). Subsequently, RNA was reversely transcribed to cDNA using the iScriptTM cDNA Synthesis Kit (Bio-Rad, USA). The qPCR reaction was performed using SsoAdvancedTM SYBR® Green Supermix Kit (Bio-Rad Company, USA) by CFX96TM Real-Time System (Bio-Rad Company, USA), with Actin7/UBC21 as reference gene (Chen et al. 2010). The primers used in qRT-PCR analysis were listed in Table.

Table Primers for qRT-PCRGene Accession Forward primer (5´ 3´) Reverse primer (5´ 3´) ReferenceBnMAPK1 JQ708034 TGGCCATCGACCTTCTTCAGA GGTAATGAAGCATCTCATTCCAC Lu et al. 2013P5CSB AF314812 CAGAAGCCACAGACTGAACTTG AAACTGCTATCAGTCACCAGCA Xue et al. 2009PLC AF108123 CTGATCGATGTTCAGAAGCAAG TCGAGGTGGAGACCGTTACTAT Das et al. 2005SCE1 NM115649.2 AGGCTTTTTCCACCCTAATGTCTATCCA ACCCTTTTCTTGTACTCAACTGCATCC Karan et al. 2012LEA4 AY572958 TGGCATGGACAAGACCAAAGCCAC ACGCGGGTGTTATGAGCGGT Park et al. 2005Actin7 EV116054 TGGGTTTGCTGGTGACGAT TGCCTAGGACGACCAACAATACT Chen et al. 2010UBC21 EV086936 CCTCTGCAGCCTCCTCAAGT CATATCTCCCCTGTCTTGAAATGC Chen et al. 2010

AcknowledgementsThis study was supported by the National Natural Science Foundation of China (31271756, 31101175) and the National “111” Project of China’s Higher Education (B12006). We are grateful to all the members of Chongqing Rapeseed Engineering & Technology Research Center, China for their help.

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(Managing editor WENG Ling-yun)