Studies On The Molecular And Biological Characteristics Of Cry1Ac Transgenic Sugarcane

Sugarcane(Saccharum spp.)is the most important sugar crop.Insect pest is one of the major constraints for sugarcane yield,quality and its cultivation period of ratoon.Stem borer is the most harmfUl pest,which can damage the sugarcane in the whole growing season and result in serious losses.Due to the complexity of sugarcane genetic background and the lack of insect-resistant gene in sugarcane gene pool,just like many other crops,which results in modern sugarcane cultivars only have poor resistance to pest.Thus,it is almost impossible to breed an insect-resistant variety by traditional cross breeding without resistant germplasms.Transgenic breeding could help us to develop insect-resistant sugarcane varieties and germplasms which can be used for cross breeding.By now,cry1Ac gene provides an effective way to control stem-borer pests in many cropssuch as corn and cotton,which has been widespread commercial applications,and their safety has been confirmed.The commercialization of genetically modified(GM)sugarcane has a good prospect:first of all,as an industrial crop and a vegetative propagation crop,sugarcanehas been classed as the lowest GM security risk crop(level 1);besides,the sucrose in commercial circulation is refined by a temperature as high as 107?.In addition,sugarcane is an indirect food,and the imported exogenous genes and expressed protein during transgenic procedure will not be detected in the sucrose refined from GM sugarcane.There is an urgent need for a borer resistance trait,the development of crylAc transgenic sugarcane has a series of study due to a lack of insect-resistant genes in sugarcane gene pool,while without approval for commercial cultivation until now.Thus,the crylAc transgenic sugarcane lines from particle bombardment are used for further study.One of the most important basic work is to establish a specific,high effective and sensitive detection technique,which can be easily used to select and verify the real transgenic sugarcane lines,and provodes a technical support for GM sugarcane breeding,tracking and supervision.In transgenic plants,the copy number of an exogenous gene can greatly affect its insert stability and inheritance,and expression level.The GM plants with relatively low copy foreign genes often showed to be stable integration and appropriate expression,while that whether sugarcane is similar or not remains to be studied.Due to the huge genome(up to 10 Gb and more than 120 chromosomes)and the complex ploidy of sugarcane,the signal of Southern blot using biotin-labelled hybridization is weak and ineffective.We developed a technology with more efficient and secure to identify the foreign gene copy number of crylAc sugarcane,which is an important meaningful for identification of the copy number in transgenic sugarcane population with low cost.CrylAc protein is the substantial foundation for insect-resistance.There is a direct correlation between insect-resistance with the crylAc protein content,and the crylAc gene expression is influenced by its structure and the environment et al.Therefore,we explored the exogenous gene expression characteristics—temporal and spatial expression of cry lAc protein.Foreign gene's insertion site in the genome can also greatly affect its heredity stability and expression level.Insertion site/flanking sequence analysis is both the foundation of the function and non-expected effect study of the exogenous gene,and the essential information for application of security release of transgenic events.Thus,we studied the insertion site/flanking sequence analysis.To explore the relationship between molecular characteristics and biological characteristics of crylAc transgenic sugarcane,based on all the collected data of about the characteristics,we conducted a correlation analysis of their yield traits(including plant heigh,stalk diameter and stalk number)and quality trait(Brix),foreign gene copy number and insect-resistance.In addition,we evaluated the effect of crylAcprotein on sugarcane rhizosphere microorganisms.Furthermore,in order to explore the borer-stress related metabolism and signal transduction pathway response mechanism differences between GM sugarcane and non-GM control,which can help us to understand the molecular basis of biological differences,RNA-seq transcriptome analysis of the typical transgenic lines(a1,a5 and 19b-4)and the non-transgenic control FN15 clone was conducted.Hence,the study has important scientific significance and practical value.This study was funded by the National Natural Science Foundation of China(31271782)and supported by the Scientific Research Foundation of Graduate School of Fujian Agriculture and Forestry University(1122YB015).The main results and conclusions of this study are as follows:1.Based on the principle of loop-mediated isothermal amplification(LAMP),specific LAMP primers were designed using the sequence of crylAc gene(GenBank:KF630361.1)and bar gene(GenBank:EU048867.1).The optimized reaction conditions of crylAc-LAMP were:5.25 mM of Mg2+,4:1 ratio inner vs outer primer,and 2.0 U of Bst DNA polymerase in a reaction volume of 25.0 ?L.The sensitivity of the crylAc.-LAMP method proved to be ten-fold higher sensitivity than that of a conventional PCR.The detection limit was 43.1 copies plasmid 1Ac0229 or 1.0 ng·?L-1 genomic DNA from crylAc transgenic sugarcane plants(21.89 crylAc copies cell-1),respectively.The optimized reaction conditions of bar-LAMP were:5.25 mM of Mg2+,6:1 ratio of inner vs outer primer,and 6.0 U of Bst DNA polymerase in a reaction volume of 25.0 ?L.The detection limit was as low as 1.0×101 copies plasmid 1Ac0229,which was ten-fold higher sensitivity than that of a conventional PCR.The high specificity and reliability of the cry1Ac-LAMP and bar-LAMP method were confirmed.In conclusion,the developed cry1Ac-LAMP and bar-LAMP assay are visual,rapid,sensitive,reliable and cost-effective for detection of the crylAc or bar specific transgenic sugarcane.More importantly,this system allowed detection of the foreign gene on-site when screening and supervision GM sugarcane without complex/expensive instruments with the results being visible to the naked eyes.This method has a very good application and promotion prospects.2.Due to the three potential endogenous reference genes,adenosine-5-phosphosulfate reductase(APRT),cyclin(CYC)and prolyl-4-hydroxylase(P4H),are high specificity and low copy number,we cloned these three genes,then reconstructed multi-endogenous reference genes standard plasmid(p1AcAPC).We established the quantitative TaqMan real-time PCR(TaqMan real-time qPCR)method based on the standard plasmid and identified the cry1Ac gene copy number of a population of transgenic sugarcane lines.The developed TaqMan real-time qPCR assay showed to highly specific,sensitive,cost-efficient and reliable for crylAcgene copy number identification.The identification results not only provide another evidence of the authenticity of transgenic sugarcane,but also lay a foundation for analysis of insert stability and inheritance,and study of systems biology.3.The results and conclusion of correlation analysis of their yield and quality traits,foreign gene copy number and insect-resistance are as following:(1)In the case of the foreign desired gene cry1Ac integrated into the sugarcane genome at a medium copy number level,it is favorable to the improvement of insect-resistance trait and tend to increase the probability of obtaining the transgenic events with a little effect on the non-target traits.This is different from a diploid plant in which a low copy integration of foreign genes is beneficial.Perhaps the main reason is due to the modern sugarcane cultivars are complex ploidy(at least octoploid,chromosome number 120 or more)and a huge genome(up to 10 Gb),low copy number of cry1Ac integrated into the sugarcane genome is ineffective for improvement of the insect-resistance trait,while high copy number of cry1Ac integrated can result in deterioration of non-objective i.e.yield and qualitytraits due to the energy consumption.Besides,we found that there is no definite linear relationship(or dose effect)between protein expression level and crylAc gene copy number.(2)The transgenic lines from recipient sugarcane variety FN15(FN15 Group)with a medium copy number of cry1Ac gene have a better phenotype of yield and quality traits,and we successfully selected one line(al)with a better sucrose yield per unit area than non-transgenic control,and two lines(a5 and a6)with a comparable sucrose yield with the control,while borer damage ratio of them was significantly lower than the control.These three lines are expected to be used directly as a cultivar or as a crossing parent to contribute their rersistance for borer resistence breeding.4.Thetemporal and spatial expression of cry1Ac protein are as following:(1)GM lines from both varieties FN15 and ROC22(ROC22 Group)trend to be the highest expression of cry1Ac protein in sugarcane leaves,then in root and the lowest in stalk.(2)GM lines from both varieties trend to be the highest expression of cryl Ac protein at tillering stage,then at maturity stage and the lowest at elongation stage.(3)The expression of cryl Ac protein in roots is complex:For FN15 Group,cryl Ac protein expression at different growing stage presents the trend that the highest expression at maturity stage,then at elongation stage and the lowest at tillering stage,while for ROC22 Group,the highest expression of cryl Ac was found at tillering stage,then at elongation stage and the lowest at maturity stage.(4)For FN15 Group,cryl Ac protein expression in lines al and a5 with medium copy number level are higher than the lines 19b-2 and 19b-2 with low copy number level at each growing stage,while not for ROC22 Group.In conclusion,crylAc protein expression in crylAc transgenic sugarcane lines presents a certain dynamic characteristics of temporal and spatial expression but with genotype difference.5.Single primer PCR,Genome walking kit(TaKaRa)and hi-Tail PCR were adopted to study the crylAcgene insertion site in GM sugarcane genome.We found that(1)Single primer PCR method has very low specificity;(2)The clone bands are too large(about 5 Kb)using Genome walking kit(TaKaRa),and the random primers(AP)is a mixture plus the patented product,which results in challenge of TA cloning and sequencing analysis,and long experimental period and high experiment cost etc.(3)Hi-Tail PCR method greatly improved the ability to obtain flanking sequence by adding AC1 and cleverly designed LAD primer.Using hi-Tail PCR method,we successfully identified the left border(LB)and right border(RB)flanking sequences of transgenic line a1 and RB flanking sequences of line a5.Besides,we found that exogenous gene via particle bombardment-mediated in crylAc transgenic sugarcane tends to integrate circular DNA i.e.mitochondria.6.Microbial community composition and evolutionary relationships of the six crylAc transgenic sugarcane lines in rhizosphere soil were investigated by PCR-DGGE(polymerase chain reaction and denaturing gradient gel electrophoresis)method.We found that soil microbial community are dynamic with which may be related to the growth periods,the seasons,or some otherenvironment factors.From the perspectiveof an overall and long-term,the cry1Actransgenic lines and non-transgenic sugarcane present a consistent effect on soil micro-ecosystem.7.Three crylAc transgenic al and a5(medium copy number level)and 19b-4(low copy number level)and their recipient sugarcane variety FN15,were sampled the+1 leaf for transcriptome analysis by RNA-seq.The results and conclusions are as follows:(1)83.50 Gb data and the total number of 668,017,350 reads were generated from 12 sugarcane samples.Trinity were used to de novo assemble a sugarcane Unigene library,which obtained a total of 65,995 Unigenes.N50 length reached 1,049 bp and the average length of the Unigenes are 715.14 bp.The above data demonstrated that the RNA-seq sequencing and assembled results were high quality.(2)The analysis of gene expression quantity and the differential expression genes were performed,along with the gene clustering pattern,functional annotation and enrichment analysis.Comparing GM lines and the non-GM line,in total of 1,067 differential expression genes were found.A total of 199 genes were up-regulated,while 473 genes were down-regulated.In all,Up/down regulated differential expression genes are abundant.(3)The results showed that,compared GM lines with the non-GM line,up-regulated differential expression genes were enriched mainly related to plant secondary metabolic pathways such as phenylalanine metabolism,glycerophospholipid metabolism,linoleic acid metabolism,flavonoid biosynthesis,diterpenoid biosynthesis etc.,and followed by the plant hormone transduction pathways;while down-regulated differential expression genes were enriched mainly related to chemical defense metabolic pathways,such as benzoxazinoid biosynthesis,and basal metabolic pathways such as fatty acid elongation etc.,and plant hormone transduction pathways and plant-pathogen interaction.The above analysis of transgenic sugarcane transcripts potential function initially revealed the molecular basis of insect-resistent trait and some other phenotypic traits of typical crylAc sugarcane lines.Further more,these pathways associated with plant defense genes,and the differential expression genes between the GM lines with medium copy number and low copy number are valuble to pay more attention in the following research.It can not only help to reveal the effect of these genes on sugarcane stress-resistence,defense and yield formation,but also can be employed to breed new GM variety with insect-resistance,high yield and quality and other objective traits.