THz Spectroscopic Detection And Gene Recognition Of Pine Wood Nematode

Sustainable development of forests is related to the global ecological environment and the survival of mankind.As an important part of global forest resources,plantation plays an important role in climate regulation,water and soil conservation.However,due to many biological instability factors,such as poor environmental resistance,biological diseases and insect pests,the productivity of plantations has been greatly reduced.Pinus species,as one of the most widely planted species in plantations,are suffering from diseases and insect pests.Bursaphelenchus xylophilus is a worldwide forest disease caused by pine wood nematode.Traditional nematode detection methods include morphology,immunology,pathology,genetics,physiology and biochemistry.However,they are often restricted by the developmental stages of plant nematodes and the detection of environmental conditions.It is difficult to achieve rapid and accurate identification.Terahertz（THz）spectroscopy,as a powerful complement to traditional detection techniques,is very sensitive to detect small differences and changes in the structure of substances,and has fingerprint characteristics reflecting the structure of compounds.Theoretical calculations show that there are a large number of intrinsic resonances（e.g.,helical twist,hydrogen bond vibration,base drift vibration mode,etc.）excited between the main chains of DNA molecules in THz band.Using THz time-domain spectroscopy technology to identify DNA bound states without fluorescent labeling of DNA fragments,the experiment is relatively simple and easy to operate.Seven kinds of biological DNA,including Bx and Bm DNA,and their main components（bases,base pairs and characteristic fragments）were studied in this paper.Using Fourier transform infrared spectroscopy,terahertz time domain spectroscopy,Raman spectroscopy and X-ray crystal diffractometer,their vibration spectra in terahertz and mid-infrared bands were obtained.Then the vibration spectra of these samples were analyzed by using the quantum mechanics method based on PBC-GEBF（generalized energy-based fragmentation approach under periodic boundary conditions）and molecular dynamics method,which laid a foundation for rapid identification of Bx and Bm.The main contents and innovations of this paper are as follows:（1）The terahertz spectra of DNA/RNA bases,base pairs and characteristic fragments were obtained and analyzed in detail.Meanwhile,the mid-infrared and Raman spectra of base crystals were studied.Five nucleic acid bases,a lab-made base-pair eutectic C:G,and two DNA characteristic fragments(5’-d（AT）₁₃-3’and 5’-d（CG）₁₃-3’,each containing 26pairs of bases)were measured by terahertz time-domain spectrometer and Fourier transform infrared spectrometer.The vibrational spectra of five nucleic acid base crystals,three base pairs containing substituents（9MA:1MT/1MC:9EG/1M5BU:9MA）and DNA/RNA characteristic fragments（5’-d（AT）₂-3’,5’-d（CG）₂-3’,5’-AUCG-3’）were calculated by using Gaussian09software,combining density functional theory and PBC-GEBF method.It is found that the theoretical and experimental spectra are in good agreement,which shows that the density functional theory and PBC-GEBF method can well describe the weak interaction between and within crystals.The vibrational modes of intracellular molecules in terahertz band originate from collective vibrations involving all atoms.The two hydrogen bonds between A:T and A:U base pairs are easy to break,and the eutectic structure is not really formed.However,due to the existence of three hydrogen bonds in the C:G eutectic structure,the hydrogen bonds are not broken during the drying process,resulting in eutectic（microcrystalline）of about several microns.Methyl and ethyl as substituents have little effect on the intensity and location of absorption peaks in molecular spectra,but halogen atom bromine as substituent has some effect on the spectra.Whatever the substituents are,they have a great influence on the vibration modes.This is because the vibration modes in terahertz band are mainly collective vibration involving all atoms or molecules.Even if the structure changes slightly,the vibration modes are greatly affected.For DNA/RNA characteristic fragments,the terahertz band vibration modes are mainly derived from the vibration or rotation of DNA/RNA skeleton,which is independent of the secondary structure of DNA/RNA.The mid-infrared and Raman theoretical spectra based on crystal structure are more consistent with the experimental spectra.（2）The terahertz spectra of seven biological DNA samples including Bx and Bm DNA were studied.The terahertz spectra of them were measured by Fourier transform infrared spectroscopy and terahertz time domain spectroscopy.It is found that the different DNA spectra measured by Fourier transform infrared spectroscopy are very similar in both THz band and Sub-THz band,and can not be distinguished by the location of absorption peaks.However,different DNA spectra measured by terahertz time-domain spectrometer have some differences in the location of absorption peaks,but because of too many absorption peaks,it is not easy to distinguish.（3）The terahertz spectra of the characteristic fragments and short sequences of ITS1region of Bx and Bm rDNA were studied.Data mining technology was used to obtain characteristic fragments（approximately 280 pairs of bases）of ITS1 region of Bx and Bm rDNA.The short sequences representing the ITS1 region of Bx/Bm rDNA were obtained by restriction endonuclease and Monte Carlo method,respectively.The samples were prepared by gene synthesis technology,and the terahertz spectra were obtained by Fourier transform infrared spectroscopy.Using AMBER software and molecular dynamics theory,the vibration spectra of all short DNA sequences were simulated.It was found that although there was no significant difference in the experimental and theoretical spectra of different short DNA sequences,the vibration modes were closely related to the internal base sequence,so the DNA of Bx and Bm could be distinguished by the vibration modes.It is also found that,to some extent,the short sequence predicted by second-order Markov chain and Monte Carlo method can represent the complete sequence as the initial structure of calculation.The longer the predicted short sequence is,the closer the calculated result is to the experimental.