Response To Heat Stress And Functional Analysis Of LsbHLH30 Gene In Lettuce(Lactuca Sativa L.)

Lettuce(Lactuca sativa L.),also known as leaf lettuce,is a variety of the genus Lettuce in the family Asteraceae,popular for its fast growth,easy cultivation,high yield and good fresh taste.Lettuce is appropriate for living in cold weather,and the suitable growth temperature is 15-25°C.Heat stress can cause dramatic changes in morphology,such as: loose plant type,bolting,and narrow and long leaves.The morphological changes seriously damage the commercial value,but greatly enhance the ability of lettuce adapt to heat stress.Currently,there is no clear understanding on the mechanism of how lettuce adaptation to heat stress.In this study,we used a heat-sensitive lettuce variety ’GRAND RAPIDS TBR’ as experimental material,by analysis morphological and physiological index,transcriptome sequencing,gene cloning and functional analysis and yeast two-hybrid technology,this study aim to uncover the physiological basis and molecular mechanism of lettuce adaptation to heat stress at the morphological,physiological and gene functional levels.The results were summarized as follows.In this study,the morphological and physiological indexes were analyzed during heat stress.The morphological changes of lettuce were not obvious from the 1st to the3 rd day.From the 5th to the 13 th day,the leaf number(LN)and bolt length(SL)of lettuce gradually increased,and the new leaves gradually became narrow and longer;The contents of chlorophyll(Chl),carotenoid(Car),proline(Pro)and soluble sugar(SS)were lower than the control from the 1st to the 5th day,and gradually higher than the control from the 7th to the 13 th day;At the same time,the activities of catalase(CAT),peroxidase(POD)and superoxide dismutase(SOD)were significantly higher than the control(P<0.01)from the 1st to the 5th day,and the activities of these enzymes gradually approached the normal temperature level from the 7th to the 13 th day;After13 days of heat stress,the shoot dry weight increased by 148.70%Further analyzing the dynamic changes of Chl content and photosynthetic parameters of lettuce,it was found that the Chl content and net photosynthetic rate(Pn)of lettuce were lower than the control from the 1st to the 3rd day after heat stress,but the difference was not significant at the 5th day,and the Chl and Pn increased gradually from the 7th to 13 th day(P<0.01);At the same time,transpiration rate(Tr)and stomatal conductance(Gs)increased gradually during heat stress.On the 13 th day,the Tr and Gs were 6.86 and 2.18 times higher than the control respectively,indicating that the photosynthesis increased gradually from the 7th day after heat stress.The transcriptome sequencing was performed at the 7th day.The results showed that the number of differentially expressed genes(DEGs)was 3146 after heat stress,of which 1843 were up-regulated and 1303 were down-regulated.KEGG analysis showed that carbon fixation,photosynthesis-antennal proteins,starch and sucrose metabolic pathways were significantly enriched,indicating that a series of changes in photosynthetic system system occurred during heat stress.Further analysis revealed that LsbHLH30 may be a key gene involved in the response to heat stress.In addition,Ls POR,a gene that regulates chlorophyll synthesis,and Lsrbc S,Ls CPN60,Ls RCA and Ls RBCX,genes that regulate photosynthesis,were significantly up-regulated(P<0.01),which may be responsible for the enhanced photosynthesis in the late stage of heat stress.LsbHLH30 was cloned and analyzed.Sequence analysis showed that LsbHLH30 contains an open reading frame of 1575 bp and encodes 228 amino acids.Physicochemical analysis revealed that LsbHLH30 has a molecular weight of 25.68 KD,a theoretical isoelectric point p I of 9.30,no transmembrane structural domain,17 phosphorylation sites,and is an unstable hydrophilic protein.CD-search analysis revealed that LsbHLH30 contains a basic Helix-Loop-Helix(b HLH)structural domain.Phylogenetic analysis showed that LsbHLH30 is most closely related to sunflower(Helianthus annuus)and cardoon(Cynara cardunculus var.scolymus)of the Asteraceae family.Subcellular localization revealed that LsbHLH30 was localized in the nucleus.Real-time fluorescence quantitative PCR analysis revealed that the expression of LsbHLH30 was higher than the control during heat stress.The function of LsbHLH30 in lettuce was verified by transgenic and yeast twohybrid.It was found that the leaves of transgenic lettuce became narrower and longer,and the stalk became longer than the control.These morphology changes were consistent with the response of lettuce to heat stress,indicating that LsbHLH30 promoted the response of lettuce to heat stress.A yeast two-hybrid screening of lettuce c DNA libraries yielded five known functional interacting proteins,including the 1,5-bisphosphate ribulose carboxylation/oxygenase small subunit Lsrbc S(Rubis CO small subunit),the stem-specific gene TSJT1(stem-specific protein),the DNA methyltransferase MET1(DNA Methyltransferase 1),the plant water channel protein PIP2-4(aquaporin PIP2-4-like)and non-specific lipid-transfer protein 1-like.Among them,Lsrbc S has the function of regulating Rubisco activity and affecting the specific binding of Rubisco to CO2/O2.It is speculated that lettuce may enhance photosynthesis through the interaction of LsbHLH30 and Lsrbc S.